Targeted Protein Degradation

ABSTRACT

The invention relates to a method of controlling the level of a polypeptide sequence comprising administering a polypeptide sequence fused to a ubiquitin targeting protein which comprises a minimal degron structural motif. In particular, the polypeptide sequence comprises a chimeric antigen receptor therefore the present invention is useful in methods of cell and gene therapy where the activity of the chimeric antigen receptor needs to be controlled.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.K. Provisional Application No.GB 1710620.4, filed 3 Jul. 2017.

FIELD OF THE INVENTION

The invention relates to methods of controlling the level and/oractivity of a heterologous protein which has been introduced into a hostcell.

BACKGROUND TO THE INVENTION

Targeted degradation of proteins has previously been achieved throughstrategies harnessing the ubiquitin proteasome system (UPS). Inparticular, Proteolysis Targeting Chimeric molecules (PROTACs) have beendescribed in the art which are heterobifunctional compounds composed ofa target protein-binding ligand and an E3 ubiquitin ligase ligand thatinduce proteasome-mediated degradation of the target protein via theirrecruitment of E3 ubiquitin ligase and subsequent ubiquitination. Suchcompounds are capable of inducing the inactivation of a target proteinupon addition to cells or administration to an animal or human, andtherefore have been proposed for the treatment of disease by removingpathogenic or oncogenic target proteins.

Chimeric antigen receptors (CARs) are artificial T-cell receptors thatare at the forefront of modern personalised therapies (Lee et al. (2012)Clin. Cancer Res., 18(10): 2780-90). They are being developed to treatcancers in patients that are resistant to conventionally availabletherapies and use a patient's own immune cells to combat the disease.The immune cells are genetically engineered ex vivo to express a CAR(CAR-T cells) specific to a tumour antigen, and the cells aresubsequently transferred back to the patient. CARs reside on surfaces ofT cells and consist of intracellular and extracellular domains which areseparated by a transmembrane domain. The extracellular domain harbours atarget binding region (e.g. a single chain variable fragment) that isdirected towards an antigen solely expressed on diseased cells. Theintracellular domain (usually CD3ζ-CD28 or CD3ζ-41BB) faces the cytosoland transmits an activation signal to the T cell after the antigen isbound to the target binding region on the surface of the cell. Activesignalling of CAR-T cells leads further to the killing of the diseasedcells.

The development of CARs has comprised three generations so far. Thefirst generation CARs comprised target binding domains attached to asignalling domain derived from the cytoplasmic region of the CD3zeta orthe Fc receptor gamma chains. First generation CARs were shown tosuccessfully retarget T cell killing to the selected target, however,they failed to provide prolonged expansion and antitumor activity invivo. The second and third generation CARs have focussed on enhancingmodified T cell survival and increasing proliferation by includingadditional signalling domains from co-stimulatory molecules, such asCD28, OX-40 (CD134) and 4-1BB (CD137).

However, a safety concern of this promising therapy has arisen throughpotential cross-reactivity to vital organs such as the lung. Indeed,during clinical trials, both on-target as well as off-target off-tumourtoxicities have been observed in patients treated with CAR-T cells andfatalities have been reported with CAR studies (Morgan et al. (2010)Mol. Ther., 18(4): 843-51). These toxicities are difficult to predict inanimal or non-primate models, and in contrast to small molecules andbiologics, CAR-T cells are living-drugs that have unique pharmacokinetic(PK) profiles and pharmacodynamic effects. Therefore, safety switchesare being developed to turn off or tune down CAR-T cell killing andallow for more controlled and safer therapies.

Suicide switches are one example of a safety switch where CAR-T cellsare further engineered to express “suicide genes” or “elimination genes”which allows selective destruction of CAR-T cells upon administration ofan external agent. For example, incorporating herpes simplex virusthymidine kinase (HSV-TK) means that administration of the prodrugganciclovir results in cell death by incorporation of GCV-triphosphateinto replicating DNA. However, the elements involved in this switch areimmunogenic and there is emerging evidence that immune responses againstHSV-TK limit the persistence of transduced cells Berger et al., (2006)Blood March 15:107(6):2294-302).

WO2017024318 describes compositions and methods for regulating chimericantigen receptor immune effector cell therapies by attaching a dTAGwhich binds a heterobifunctional compound which, in turn, leads toubiquitination.

In order for cellular therapies to be become more widely adopted, thereis still a need in the art to develop methods for controlling thesetherapies to ensure that any adverse events can be prevented.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a methodof controlling the level of a polypeptide sequence comprising:

a) administering a fusion protein comprising said polypeptide sequenceand a ubiquitin targeting protein consisting of less than 135 aminoacids in length which comprises the hairpin motif of a cereblon bindingsite, and

b) controlling the level of the polypeptide sequence by administering acompound which mediates binding of the ubiquitin targeting protein andcereblon.

In another aspect of the invention there is provided a method ofcontrolling the level of a polypeptide sequence comprising:

a) administering a fusion protein comprising:

-   -   A-B

wherein A is a polypeptide sequence; and

wherein B is a ubiquitin targeting protein consisting of less than 135amino acids in length comprising a structural motif which when alignedhas a set of structural coordinates within about 6.0 Å of the rmsd ofthe backbone atoms between each of the amino acid residues as listed inTable 1, and wherein the structural motif comprises a glycine residue atthe position which corresponds to GLY56 of Table 1; and

b) controlling the level of the polypeptide sequence by administering acompound which mediates binding of a) the ubiquitin targeting proteinand b) a ubiquitin ligase in a manner that brings the polypeptidesequence into proximity of the ubiquitin ligase, wherein the polypeptidesequence, in the presence of the compound, is capable of beingubiquitinated.

According to a further aspect of the invention, there is provided achimeric antigen receptor (CAR) comprising:

an extracellular ligand binding domain;

a transmembrane domain;

an intracellular signalling domain; and,

a ubiquitin targeting protein as described herein, which is capable ofbeing bound by ubiquitin ligase in the presence of a compound.

According to a further aspect of the invention, there is provided afusion protein comprising a polypeptide sequence consisting of less than135 amino acids in length, which comprises the hairpin motif of acereblon binding site.

In another aspect of the invention there is provided a fusion proteincomprising

-   -   A-B

wherein A is a polypeptide sequence; and

wherein B is a ubiquitin targeting protein which consists of a sequenceselected from the group consisting of: SEQ ID NOs: 6-14.

DESCRIPTION OF THE FIGURES

FIG. 1. Schematic organisation of chimeric antigen receptor constructthat incorporates a degron element.

FIG. 2. Plasmid map encoding GFP fused to degron signal derived fromhuman Ikaros 1 protein (Uniprot Q13422).

FIG. 3. Effect of lenalidomide treatment on the expression levels ofGreen Fluorescent Protein (GFP) fused with degrons in HEK293T cells. (A)Table with the values of median fluorescence intensity of GFP positivecells treated with increasing amount of lenalidomide. Constructsincluded in the figure differ only in the length of the(Glycine-Serine)×N linker between GFP and the degron sequence, withCONSTRUCT 1 N=1, CONSTRUCT 2 N=3 and CONSTRUCT 3 N=5. (B) Medianfluorescence intensity (MFI) values relative to untreated (nolenalidomide) values.

FIG. 4. Plasmid map encoding chimeric antigen receptor (CAR) constructused as a control for lenalidomide induced CAR degradation. ZsGreen usedas a reporter for transfection/transduction.

FIG. 5. Effect of lenalidomide treatment on the expression levels ofanti-BCMA CARs fused with degrons in Jurkat cells. (A) Medianfluorescence intensity of anti-BCMA-CAR constructs in transfected(ZsGreen positive) Jurkat cells after treatment with DMSO or 10 μMlenalidomide. CONSTRUCT 4 represents a CAR without a degron signal whileCONSTRUCT 5 contains that same elements of CONSTRUCT 4 plus sequencesderived from human Ikaros 3 (Uniprot Q9UKT9) protein. (B) Flow cytometryhistograms of the Jurkat cells in (A), showing the effect of DMSO and 10μM lenalidomide on anti-BCMA CAR expression.

FIG. 6. Effect of lenalidomide treatment on the expression levels ofanti-BCMA CARs fused with degrons in primary T-cells. (A) Medianfluorescence intensity of anti-BCMA CAR in transduced (ZsGreen positive)T-cells cells after treatment with DMSO or 10 μM lenalidomide. CONSTRUCT4 represents a CAR without a degron signal while CONSTRUCT 5 containsthat same elements of CONSTRUCT 4 plus sequences derived from humanIkaros 3 (Uniprot Q9UKT9) protein. Data are representative of threebiological repeats. (B) Flow cytometry histograms of the T-cells in (A),showing the effect of DMSO and 10 μM lenalidomide on anti-BCMA CARexpression.

FIG. 7. Effect of lenalidomide treatment on the cytokine release fromprimary T-cells expressing anti-BCMA CARs fused with degrons. PrimaryT-cells were transduced with CONSTRUCT 4 or 5, and co-cultured withBCMA-expressing ARH-77-10B5 cells in the presence of DMSO or 10 μMlenalidomide. CONSTRUCT 4 represents a CAR without a degron signal whileCONSTRUCT 5 contains that same elements of CONSTRUCT 4 plus sequencesderived from human Ikaros 3 (Uniprot Q9UKT9) protein. Data arerepresentative of three biological repeats.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is an essential structural motif required for effectiveubiquitination of a target protein. This can be used to control thelevel and/or activity of a heterologous protein administered to a targetcell, especially when the heterologous protein is a therapeutic proteinwhich needs to be tightly controlled in order to avoid the risk of anyadverse effects. Minimising the size of the motif required for effectiveubiquitination has advantages, especially if the heterologous protein isintroduced via a viral vector where space is a premium.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart (e.g., in cell culture, molecular genetics, nucleic acid chemistry,hybridization techniques and biochemistry). Standard techniques are usedfor molecular, genetic and biochemical methods (see generally, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed. (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel etal., Short Protocols in Molecular Biology (1999) 4^(th) Ed, John Wiley &Sons, Inc. which are incorporated herein by reference in their entirety)and chemical methods. All patents and publications referred to hereinare incorporated by reference in their entirety.

The term “comprising” encompasses “including” or “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “consisting essentially of” limits the scope of the feature tothe specified materials or steps and those that do not materially affectthe basic characteristic(s) of the claimed feature.

The term “consisting of” excludes the presence of any additionalcomponent(s).

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% fromthe specified value.

The term “safety switch” refers to a biochemical mechanism that can beactivated on demand in order to control a biological process which cancause harm. Safety switches can be used with CAR-T therapies so thatthey can be controlled externally (i.e. via administration from outsideof the cell) in order to enhance the safety of the gene therapy.

The term “chimeric antigen receptors” (“CARs”) as used herein, refers toan engineered receptor which consists of an extracellular target bindingdomain (which is usually derived from a monoclonal antibody or fragmentthereof), optionally a spacer region, a transmembrane region, and one ormore intracellular effector domains. CARs have also been referred to aschimeric T cell receptors or chimeric immunoreceptors (CIRs). CARs aregenetically introduced into hematopoietic cells, such as T cells, toredirect specificity for a desired cell-surface antigen. More recentlyCARs have also been introduced to non-immune cells (Kojima et al., 2018Nature Chem. Bio. January; 14(1):42-49).

References to “CAR signalling” refer to signalling through thesignalling domain of the CAR which results in immunomodulatory cellactivation (e.g. triggering target cell killing and T cell activation).

The term “T cell receptor” (“TCR”) as used herein, refers to thereceptor present on the surface of T cells which recognises fragments ofantigen as peptides bound to major histocompatibility complex (MHC)molecules. Native TCRs exist in αβ and γδ forms, which are structurallysimilar but exist in different locations and are thought to havedifferent functions. The extracellular portion of the TCR has twoconstant domains and two variable domains. The variable domains containpolymorphic loops which form the binding site of the TCR and areanalogous to complementarity determining regions (CDRs) in antibodies.In the context of gene therapies, the TCR is usually geneticallymodified to change or improve its antigen recognition, therefore in oneembodiment, the TCR is genetically modified. For example, WO01/055366and WO2006/000830, which are herein incorporated by reference, describeretrovirus-based methods for transfecting T cells with heterologousTCRs.

The term “target binding domain” as used herein is defined as an oligo-or polypeptide that is capable of binding a specific target, such as anantigen or ligand. In particular, the target may be a cell surfacemolecule. For example, the target binding domain may be chosen torecognise a target that acts as a cell surface marker on pathogeniccells, including pathogenic human cells, associated with a particulardisease state. The target binding domain may be, for example, any typeof protein which binds to an antigen.

The term “spacer region” as used herein, refers to an oligo- orpolypeptide that functions to link the transmembrane domain to thetarget binding domain. This region may also be referred to as a “hingeregion” or “stalk region”. The size of the spacer can be varieddepending on the position of the target epitope in order to maintain anoptimum distance for activation of the immune synapse (e.g. 14 nm) uponCAR:target binding.

The term “domain” refers to a folded protein structure which retains itstertiary structure independent of the rest of the protein. Generally,domains are responsible for discrete functional properties of proteinsand in many cases, may be added, removed or transferred to otherproteins without loss of function of the remainder of the protein and/orof the domain.

The term “transmembrane domain” as used herein refers to a domain whichtraverses the cell membrane.

The term “intracellular effector domain” as used herein refers to thedomain in the CAR which is responsible for intracellular signallingfollowing the binding of the target binding domain to the target. Theintracellular effector domain is responsible for the activation of atleast one of the normal effector functions of the immune cell in whichthe CAR is expressed. For example, the effector function of a T cell canbe a cytolytic activity or helper activity including the secretion ofcytokines.

The term “antibody” is used herein in the broadest sense to refer tomolecules with an immunoglobulin-like domain (for example IgG, IgM, IgA,IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric,human, humanised, multispecific antibodies, including bispecificantibodies, and heteroconjugate antibodies; a single variable domain(e.g., VH, VHH, VL, domain antibody (dAb)), antigen binding antibodyfragments, Fab, F(ab′)₂, Fv, disulphide linked Fv, single chain Fv,disulphide-linked scFv, diabodies, TANDABS, etc., and modified versionsof any of the foregoing.

The term “single variable domain” refers to a folded polypeptide domaincomprising sequences characteristic of antibody variable domains. Ittherefore includes complete antibody variable domains such as VH, VHHand VL and modified antibody variable domains, for example, in which oneor more loops have been replaced by sequences which are notcharacteristic of antibody variable domains, or antibody variabledomains which have been truncated or comprise N- or C-terminalextensions, as well as folded fragments of variable domains which retainat least the binding activity and specificity of the full-length domain.A single variable domain is capable of binding an antigen or epitopeindependently of a different variable region or domain. A “domainantibody” or “dAb” may be considered the same as a “single variabledomain”. A single variable domain may be a human single variable domain,but also includes single variable domains from other species such asrodent (for example, as disclosed in WO 00/29004), nurse shark andCamelid VHH dAbs. Camelid VHH are immunoglobulin single variable domainpolypeptides that are derived from camelid species including bactrianand dromedary camels, llamas, vicugnas, alpacas, and guanacos, whichproduce heavy chain antibodies naturally devoid of light chains. SuchVHH domains may be humanised according to standard techniques availablein the art, and such domains are considered to be “single variabledomains”. As used herein VH includes camelid VHH domains.

For the avoidance of doubt, it will be understood that the terms“polynucleotide”, “nucleotide” and “nucleic acid” are usedinterchangeably herein.

For the avoidance of doubt, it will be understood that the terms“polypeptide”, “oligopeptide” “peptide” and “amino acid” are usedinterchangeably herein.

For the avoidance of doubt, it will be understood that the terms“structural motif” and “hairpin motif” are used interchangeably herein.

References to “fusion protein” refer to a protein translated from afusion gene, which is created by joining parts of two differentgenes/nucleic acid sequences. The polypeptide sequence A and ubiquitintargeting protein B are associated with one another, preferably bygenetic fusion (i.e. the fusion protein is generated by translation of anucleic acid in which a polynucleotide encoding all or a portion of A isjoined in-frame with a polynucleotide encoding all or a portion of B) orchemical conjugation to one another.

References to “functional fragments” refer to fragments of the full,wild-type sequences which still retain the binding function of the wildtype protein from which they are derived (e.g. functional fragments ofthe ubiquitin targeting protein still enable binding to the compoundwhich mediates the interaction with the ubiquitin ligase). Fragments maysuitably comprise at least 10 amino acids in length, for example 25, 50,75, 80, 90, 100, 110, 120 or 130 amino acids in length. Fragments mayalso comprise a C-terminal truncation, or an N-terminal truncation.

References to “functional variants” include variants with similar aminoacid or nucleotide sequences to the original (e.g. wild-type) sequences,but with one or more amino acid or nucleotide changes that result in avariant which still retains the function of the original protein fromwhich they are derived. For example, a functional variant of theubiquitin targeting protein described herein include variants that stillfacilitate sufficient binding to the compound which enablesubiquitination of the polypeptide sequence via the ubiquitin ligase.

“Affinity” is the strength of binding of one molecule, e.g. the targetbinding protein, to another, e.g. its target antigen, at a singlebinding site. The binding affinity of a target binding protein to itstarget may be determined by equilibrium methods (e.g. enzyme-linkedimmunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics(e.g. BIACORE analysis).

Sequence identity as used herein is the degree of relatedness betweentwo or more amino acid sequences, or two or more nucleic acid sequences,as determined by comparing the sequences. The comparison of sequencesand determination of sequence identity may be accomplished using amathematical algorithm; those skilled in the art will be aware ofcomputer programs available to align two sequences and determine thepercent identity between them. The skilled person will appreciate thatdifferent algorithms may yield slightly different results.

Thus, the “percent identity” between a query nucleic acid sequence and asubject nucleic acid sequence is the “Identities” value, expressed as apercentage, that is calculated by the BLASTN algorithm when a subjectnucleic acid sequence has 100% query coverage with a query nucleic acidsequence after a pair-wise BLASTN alignment is performed. Such pair-wiseBLASTN alignments between a query nucleic acid sequence and a subjectnucleic acid sequence are performed by using the default settings of theBLASTN algorithm available on the National Center for BiotechnologyInstitute's website with the filter for low complexity regions turnedoff. Importantly, a query nucleic acid sequence may be described by anucleic acid sequence identified in one or more claims herein.

Similarly, the “percent identity” between a query amino acid sequenceand a subject amino acid sequence is the “Identities” value, expressedas a percentage, that is calculated by the BLASTP algorithm when asubject amino acid sequence has 100% query coverage with a query aminoacid sequence after a pair-wise BLASTP alignment is performed. Suchpair-wise BLASTP alignments between a query amino acid sequence and asubject amino acid sequence are performed by using the default settingsof the BLASTP algorithm available on the National Center forBiotechnology Institute's website with the filter for low complexityregions turned off. Importantly, a query amino acid sequence may bedescribed by an amino acid sequence identified in one or more claimsherein.

The query sequence may be 100% identical to the subject sequence, or itmay include up to a certain integer number of amino acid or nucleotidealterations as compared to the subject sequence such that the % identityis less than 100%. For example, the query sequence is at least 50, 60,70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subjectsequence. Such alterations include at least one amino acid deletion,substitution (including conservative and non-conservative substitution),or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the query sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids or nucleotides in the query sequence or in one or morecontiguous groups within the query sequence.

The term “ubiquitin targeting protein” refers to theprotein/domain/fragment which is capable of inducing ubiquitination ofthe polypeptide sequence by binding ubiquitin ligase in the presence ofa compound.

The term “ubiquitin ligase”, also known as E3 ligase, refers to a familyof proteins that facilitate the transfer of ubiquitin-alone or incomplex- to a specific substrate protein, therefore targeting thesubstrate protein for degradation. For example, cereblon is part of anE3 ubiquitin ligase complex Cul4A/B that in combination with an E2ubiquitin-conjugating enzyme causes the attachment of ubiquitin to alysine on a target protein, and subsequently targets the specificprotein for degradation by the proteasome. The ubiquitin ligase may beinvolved in polyubiquitination such that more than one ubiquitin isattached to the target protein. For example, a second ubiquitin isattached to the first ubiquitin; a third is attached to the second, andso forth. Polyubiquitination can mark proteins for degradation by theproteasome.

The term “ubiquitination site” refers to the amino acid residue, inparticular, a lysine residue to which ubiquitin is attached. In order tomake a chain, ubiquitin itself contains a ubiquitination site. Differentlysines on ubiquitin can be targeted by an E3 to make chains, but themost common lysine is Lys48. This lysine-48 may be used to makepolyubiquitin, which is then recognized by the proteasome.

The term “autologous” as used herein, refers to cells from the samesubject. The term “allogeneic” as used herein, refers to cells of thesame species that differ genetically to the cell in comparison.

The terms “individual”, “subject” and “patient” are used hereininterchangeably. In one embodiment, the subject is a mammal, such as aprimate, for example a marmoset or monkey, or a human. In a furtherembodiment, the subject is a human.

The term “pharmaceutical composition” refers to a composition formulatedin pharmaceutically-acceptable or physiologically-acceptable solutionsfor administration to a cell or subject. The compositions of theinvention may be administered in combination with other agents as well,provided that the additional agents do not adversely affect the abilityof the composition to deliver the intended therapy.

The term “cancer” (sometimes also referred to as “neoplasia”) refers toa disease caused by an uncontrolled division of abnormal cells in a partof the body. The uncontrolled division can often result in a mass,commonly referred to as a “tumour” or “neoplasm”. The term “tumourassociated antigen” or “tumour antigen” as used herein, refers to anantigen expressed on a tumour cell. This antigen may be uniquely ordifferentially expressed on a tumour cell when compared to a normal,i.e. non-cancerous, cell.

The invention described herein may also be used in methods of treatmentof a subject in need thereof. Treatment can be therapeutic, prophylacticor preventative. Treatment encompasses alleviation, reduction, orprevention of at least one aspect or symptom of a disease andencompasses prevention or cure of the diseases described herein.

Methods of Controlling Protein Levels

Structural conservation of the cereblon binding site has been observedfor CK1 and GSTP1. For Ikaros 1 and 3, the structural conservation hasbeen inferred by homology to structurally characterized proteins such asEos (Ikaros 4, PDB entry 2MA7) or the zinc finger protein from PDB entry2113. The present inventors have proposed fusing this conservedstructural motif to a heterologous protein in order to provide a methodof controlling the level and/or activity of said protein. In contrast tocurrent PROTAC methods used in the art, the present method involvesfusing a ubiquitin targeting protein (which may also be referred to as aubiquitin targeting domain) directly to the protein to be controlled sothat an external compound can simply be added in order to induce bindingto ubiquitin ligase which results in ubiquitination.

Furthermore, by using the minimal degron required, there are potentialproduction advantages. For example, vectors are often size limited,therefore it is helpful to have a small ubiquitin targeting protein tomaximise the amount of space available to encode other polypeptidesequences.

A minimal degron is also less likely to interfere with the activity ofthe heterologous protein.

Therefore, according to a first aspect of the invention, there isprovided a method of controlling the level of a polypeptide sequencecomprising:

a) administering a fusion protein comprising said polypeptide sequenceand a ubiquitin targeting protein consisting of less than 135 aminoacids in length which comprises the hairpin motif of a cereblon bindingsite, and

b) controlling the level of the polypeptide sequence by administering acompound which mediates binding of the ubiquitin targeting protein andcereblon.

In another aspect of the invention there is provided a method ofcontrolling the level and/or activity of a polypeptide sequencecomprising:

a) administering a fusion protein comprising:

-   -   A-B

wherein A is a polypeptide sequence; and

wherein B is a ubiquitin targeting protein consisting of less than 135amino acids in length comprising a structural motif which when alignedhas a set of structural coordinates within about 6.0 Å of theroot-mean-square deviation (rmsd) of the backbone atoms between each ofthe amino acid residues as listed in Table 1, and wherein the structuralmotif comprises a glycine residue at the position which corresponds toGLY56 of Table 1; and

b) controlling the level of the polypeptide sequence by administering acompound which mediates binding of a) the ubiquitin targeting proteinand b) a ubiquitin ligase in a manner that brings the polypeptidesequence into proximity of the ubiquitin ligase, wherein the polypeptidesequence, in the presence of the compound, is capable of beingubiquitinated.

Without being bound by theory, once the polypeptide sequence has beenubiquitinated it can be degraded by a proteasome or attachment ofubiquitin (in particular a polyubiquitin chain) causes the polypeptidesequence to be sterically inhibited. Thus, in one embodiment, thepolypeptide sequence is capable of being ubiquitinated and then degradedby a proteasome. In an alternative embodiment, the polypeptide sequenceis capable of being ubiquitinated and then sterically inhibited.

Ubiquitin is a small (about 8.5 kDa) protein that has been found in mosttissues of eukaryotic organisms. The addition of ubiquitin to asubstrate protein is called ubiquitination or ubiquitylation.Ubiquitination can affect proteins in many ways, including signallingfor their degradation via the proteasome. Ubiquitin is covalentlycoupled to a substrate lysine by activity of an E1 (ubiquitin activatingenzyme), E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin ligase)enzyme cascade. A E3 ubiquitin RING ligase is a protein that recruits anE2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin,therefore E3 ubiquitin RING ligases interact with both the targetprotein and the E2 enzyme. One example of an E3 ubiquitin ligase iscereblon (CRBN) which interacts with damaged DNA binding protein 1(DDB1), Cullin 4 (CUL4) and RING-box protein 1 (RBX1) to form thecomplex CUL4-RBX1-DDB1-CRBN. This complex then ubiquitinates the proteinsubstrate which is subsequently degraded by proteasomes.

The method of the invention allows the level (e.g. the intracellular,extracellular or membrane levels) and/or activity of a polypeptidesequence to be controlled by fusing it to domain that allows fortargeted ubiquitination upon introduction of a mediating compound.Addition of the compound can cause the level of polypeptide sequence tobe reduced because the compound will induce ubiquitination of thepolypeptide by ubiquitin ligase and could lead to subsequentdegradation. The change in level and/or activity can be measured usingmethods known in the art.

It will be understood that references to “ubiquitin ligase” as usedherein includes ubiquitin ligases and/or a protein which is part of theligase complex e.g. cereblon. Therefore, in one embodiment, theubiquitin ligase is cereblon.

Certain protein substrates have been shown to interact with E3 ubiquitinligases through immunomodulatory drugs (e.g. IMiDs), such asthalidomide. These protein substrates include Ikaros3, Ikaros1, GSTP1and CK1 alpha. Recently, ZFP91 was shown to be an IMiD-dependentsubstrate of cereblon (see An et al. (2017) Nat. Commun. 8: 15398).

A “minimal” degron is described herein, in the form of a small proteindomain (around 30 amino acids) that can be added to a protein ofinterest for degradation. In the presence of an IMiD compound,degradation will be induced. However, the degron is not defined by thelinear peptide sequence, but rather the geometric arrangement of threebackbone hydrogen bond acceptors at the apex of a turn (positions i,i+1, and i+2), with a glycine residue at a key position (i+3). Thisgeometric arrangement forms a hairpin motif.

When the hairpin motifs from three cereblon substrates were superimposedusing main chain atoms, the overall rmsd was found to be around 2.0 Å.In particular, the closest structural overlap was seen around a centralglycine residue which was conserved between all three motifs. TheRamachandran angles were measured in PDB entry 2113 as a structuralrepresentative of Ikaros3 ZFN (with percentage identity of 50%) andfound to be as follows:

TABLE 1 Ramachandran angles of cereblon binding hairpin motifs Ikaros3(2I13) Residue Psi (ψ) Phi (φ) LYS 51 151.5 −102.8 CYS 52 127.8 −83.5PRO 53 −7.5 −66 GLU 54 −52.5 −111.7 CYS 55 −16.9 −105.2 GLY 56 −4.9 91.2LYS 57 127.9 −60.6 SER 58 148.5 −97.5 PHE 59 155.5 −131.2 SER 60 −32.1−73.3

In one embodiment, the structural coordinates are within about 5.0 Å,such as about 4.5 Å, of the rmsd of the backbone atoms between each ofthe amino acid residues as listed in Table 1.

References to “Ramachandran angles” and “rmsd” are well known to aperson skilled in the art. Ramachandran angles are used to describe theconformation of the peptide main chain. Two torsion angles in thepolypeptide chain, also called Ramachandran angles, describe therotations of the polypeptide backbone around the bonds between N-Cα(called Phi, φ) and Cα-C (called Psi, ψ), see Ramachandran et al. (1963)J. Mol. Biol., 7:95-99. Therefore, φ and ψ describe the rotation of thepolypeptide chain around the two bonds on both sides of the Cα atom. Theangles can be plotted in a Ramachandran plot to provide a way to viewthe distribution of torsion angles in a protein structure.

In bioinformatics, the root-mean-square deviation (rmsd) of atomicpositions is the measure of the average distance between the atoms(usually the backbone atoms) of superimposed proteins. Typically, rmsdis used as a quantitative measure of similarity between two or moreprotein structures. The lower rmsd, the higher the similarity betweentwo structures.

Determination of whether a structural motif falls within the rmsd of 5 Åfrom the motif shown in Table 1 would be well known to a person skilledin the art. Such calculation is carried out by the optimisation of thesuperposition of the coordinates of carbon alpha atoms of a givenstructural motif and the coordinates of those atoms in the structuralmotif contained in Table 1. Such optimisation will produce the minimumaverage rmsd of the two structural motifs. In the superposition referredto in the scope of this claim, carbon atoms on residues on both sides ofthe central glycine are to be considered equivalent and their rmsdvalues are the ones to be included in the calculation. Suitable proteinmodelling computer programs may be used in these calculations and areknown in the art, for example Molecular Operating Environment (MOE),2013.08; Chemical Computing Group ULC.

In one embodiment, the ubiquitin targeting protein is derived from azinc finger protein.

In one embodiment, the structural motif comprises an amino acid sequenceof SEQ ID NO: 1:

-   -   X₁X₂X₃X₄X₅GX₇X₈X₉X₁₀

wherein X represents any amino acid or is absent. In a furtherembodiment, the structural motif of SEQ ID NO: 1 additionally comprisesone, two, three or more amino acids. In a yet further embodiment, thestructural motif comprises an amino acid sequence of SEQ ID NO: 15:

-   -   X₁X₂X₃X₄X₅GX₇X₈X₉X₁₀X₁₁X₁₂

wherein X represents any amino acid or is absent.

In one embodiment, one or more of the following apply:

X₁ represents valine (V), isoleucine (I) or is absent;

X₂ represents aspartic acid (D), asparagine (N) or is absent;

X₃ represents lysine (K), isoleucine (I) or is absent;

X₄ represents glutamine (Q), lysine (K) or threonine (T);

X₅ represents cysteine (C), serine (S) or asparagine (N);

X₇ represents alanine (A) or glutamic acid (E);

X₈ represents serine (S), lysine (K) or glutamic acid (E);

X₉ represents phenylalanine (F), serine (S) or valine (V);

X₁₀ represents threonine (T), lysine (K) or alanine (A);

X₁₁ represents glutamine (Q), threonine (T) or valine (V); and/or

X₁₂ represents lysine (K) or arginine (R).

In one embodiment, the structural motif is derived from a mammalianprotein, such as a human protein.

In one embodiment, the structural motif is derived from Ikaros3,Ikaros1, Casein Kinase 1 alpha (CK1 alpha), Eukaryotic peptide chainrelease factor GTP-binding subunit ERF3A (GSTP1) or Zinc Finger Protein91 (ZFP91). In a further embodiment, the structural motif is obtainedfrom the Zinc Finger Nuclease 2 motif of Ikaros3. In an alternativeembodiment, the structural motif is obtained from CK1 alpha. Sequenceinformation for these proteins is available in the art, for example seeUniProt ID numbers: Q9UKT9 for Ikaros3, P15170 for GSPT1, Q96JP5 forZFP91 and P48729 for CK1 alpha. Structural information for theseproteins is also available in the art, for example see PDB entries: 2MA7or 2113 which can be used as a structural representative for Ikaros3,5HXB for GSTP1 and 5FQD for CK1 alpha.

In one embodiment, the structural motif comprises a sequence with atleast 80%, 85%, 90%, 95% or 97% homology/identity to a sequence selectedfrom the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4and SEQ ID NO: 5. In a further embodiment, the structural motifcomprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 99%identity to a sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO: 4. In a yet further embodiment, thestructural motif comprises a sequence with at least 80%, 85%, 90%, 95%,97% or 99% identity to an amino acid sequence of SEQ ID NO: 2.

In one embodiment, the structural motif comprises a sequence selectedfrom SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or a variantthereof. For example, the variant sequence may have up to 5, 4, 3, 2 or1 amino acid substitution(s), addition(s) or deletion(s). Typically, thevariation is a substitution, particularly a conservative substitution.The variant sequence may substantially retain the biologicalcharacteristics of the unmodified protein.

In one embodiment, the structural motif comprises a sequence selectedfrom the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4and SEQ ID NO: 5. In a further embodiment, the structural motifcomprises a sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO: 4. In a yet further embodiment, thestructural motif comprises an amino acid sequence of SEQ ID NO: 2.

In one embodiment, the ubiquitin targeting protein is a polypeptidesequence consisting of less than 130 amino acids in length, such as lessthan 125, 120, 115, 110, 105, 100, 90, 80, 70, 60 or 50 amino acids inlength. In a further embodiment, the ubiquitin targeting protein is apolypeptide sequence consisting of less than 100 amino acids in length.

In one embodiment, the ubiquitin targeting protein comprises aubiquitination bait, i.e. a sequence which comprises a lysine residuewhich can act as a ubiquitination site. This embodiment can be used, forexample, when the polypeptide sequence does not itself contain a sitesuitable for ubiquitination, therefore a ubiquitination bait containinga ubiquitination site can be included within the ubiquitin targetingprotein. In one embodiment, the ubiquitination site comprises a lysineresidue.

In one embodiment, the ubiquitination bait comprises a sequence with atleast 80%, 85%, 90%, 95% or 97% homology/identity to SEQ ID NO: 16. Inone embodiment, the ubiquitination site comprises SEQ ID NO: 16 or avariant thereof. For example, the variant sequence may have up to 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s), addition(s) ordeletion(s). Typically, the variation is a substitution, particularly aconservative substitution. The variant sequence may substantially retainthe biological characteristics of the unmodified protein.

In one embodiment, the ubiquitin targeting protein consists of asequence selected from the group consisting of: SEQ ID NOs: 6-14 orfunctional fragments or functional variants thereof.

In one embodiment, the ubiquitin targeting protein consists of asequence selected from the group consisting of: SEQ ID NOs: 6-14, 27 orfunctional fragments or functional variants thereof.

In one embodiment, the ubiquitin targeting protein consists of asequence with at least 80%, 85%, 90%, 95%, 97% or 99% identity to asequence selected from the group consisting of: SEQ ID NOs: 6-14. In oneembodiment, the ubiquitin targeting protein consists of a sequenceselected from the group consisting of: SEQ ID NOs: 6-14.

In one embodiment, the structural motif is derived from Ikaros3 andcomprises SEQ ID NOs: 6, 7, 8, 9 or 10. In an alternative embodiment,the structural motif is derived from GSTP1 and comprises SEQ ID NOs: 11or 12. In another alternative embodiment, the structural motif isderived from CK1 alpha and comprises SEQ ID NOs: 13 or 14.

If the structural motifs according to the invention are isolated fromlarger IMiD-dependent cereblon substrates, they may contain amino acidresidues which form hydrophobic patches which can induce aggregation.Such patches can be mutated in order to attenuate any aggregation. Aminoacids that are considered hydrophobic include: alanine (Ala), cysteine(Cys), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine(Phe), methionine (Met), tyrosine (Tyr) and tryptophan (Trp). If theseresidues are present in the hydrophobic patch they could be mutated toanother type of amino acid residue, such as a neutral or hydrophilicresidue, for examples serine (Ser), threonine (Thr), histidine (His),arginine (Arg), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu),asparagine (Asn), glycine (Gly) proline (Pro) or glutamine (Gin), inorder to minimise aggregation.

For example, the structure of GSTP1 in complex with cereblon and CC-885(a cereblon modulator compound) was reported by Matyskiela et al.((2016) Nature 535(7611): 252-257) and deposited by PDB entry 5HXB withdata to 3.6 Å resolution. The C-terminal domain of GSTP1 (residues388-499) could be isolated and used a degron as it contains the cereblonbinding motif and an ubiquitination site. Therefore, in one embodiment,the structural motif may comprise SEQ ID NO: 11 (i.e. residues 388 to499 of GSTP1).

The area exposed by removing the N-terminal domain is 385 Å² and theresidues involved are:

TABLE 2 Exposed residues of GSTP1 upon removal of N-terminal domainSurface Area Surface Area Residues in in full length as C terminalHydrophobic Residue Position (Å2) domain (Å2) Patch HIS 388 53.3 91.1SER 389 30.1 47.9 HIS 417 47.6 80.5 x THR 418 21.1 75.4 CYS 419 12 34.1ILE 420 75.3 76.3 THR 462 44.7 62.5 ILE 463 25.4 32.5 CYS 464 9.8 62.7LEU 465 9.5 10.8 GLU 466 10.4 31.3 PHE 471 36.5 72.9 x GLN 473 41.1 73.4x MET 474 16.2 67.3 xwhere the residues in bold are involved in a hydrophobic patch of 90 Å².Therefore, residues His417, Phe471, Gln473 and/or Met474 can be mutatedto polar or neutral residues, such as serine, in order to reduce thehydrophobic patch. In one embodiment, the structural motif comprises avariant of SEQ ID NO: 11 wherein residues His417, Phe471, Gln473 and/orMet474 are mutated to another amino acid, such as a neutral amino acid.

In one embodiment, the structural motif may comprise SEQ ID NO: 12 (i.e.residues 388 to 499 of GSTP1 with mutations F471S and M474S). In thisembodiment, Gly437 represents the key central glycine and Lys493 acts asthe ubiquitination site.

In another example, the structure of CK1 alpha in complex with cereblonand lenalidomide was reported by Petzold et al. ((2016) Nature532(7597): 127-130) and deposited by PDB entry 5FQD with data to 2.45 Åresolution. Again, removal of the N-terminal and/or C-terminal domainswill expose hydrophobic patches. In particular, residues Leu63, Leu67and Ile73 are involved in a hydrophobic patch, therefore these could becandidates for mutations in order to reduce the area of the hydrophobicpatch. Therefore, in one embodiment, the structural motif comprises avariant of SEQ ID NO: 13 wherein residues Leu63, Leu67 and/or Ile73 aremutated to another amino acid, such as a neutral amino acid. In afurther embodiment, the structural motif may comprise SEQ ID NO: 14(i.e. residues 8 to 94 of CK1 alpha with mutations L63H, L67Q and I73Q).In this embodiment, Gly40 represents the key central glycine and Lys62and Lys65 act as ubiquitination sites.

In one embodiment, A is a polypeptide sequence that encodes a mammalianprotein. In a further embodiment, the mammal is a human or a mouse.

In one embodiment, A encodes a polypeptide sequence that is notnaturally found in the cell (i.e. a non-native protein).

In one embodiment, A encodes a chimeric antigen receptor (CAR) or a Tcell receptor (TCR).

In one embodiment, the polypeptide sequence controlled by administeringa compound which mediates binding of the ubiquitin targeting protein andcereblon encodes a mammalian protein. In a further embodiment, themammal is a human or a mouse.

In one embodiment, the polypeptide sequence controlled by administeringa compound which mediates binding of the ubiquitin targeting protein andcereblon encodes a protein not naturally found in the cell (i.e. anon-native protein).

In one embodiment, the polypeptide sequence controlled by administeringa compound which mediates binding of the ubiquitin targeting protein andcereblon is a transmembrane protein.

In one embodiment, the polypeptide sequence controlled by administeringa compound which mediates binding of the ubiquitin targeting protein andcereblon encodes a chimeric antigen receptor (CAR) or a T cell receptor(TCR).

Fusion proteins may be prepared using standard techniques known in theart, including chemical conjugation. For example, DNA sequences encodingthe polypeptide components (A and B) may be assembled separately, andligated into an appropriate expression vector. The 3′ end of the DNAsequence encoding one polypeptide component is ligated, with or withouta peptide linker, to the 5′ end of a DNA sequence encoding the secondpolypeptide component so that the reading frames of the sequences are inphase. This permits translation into a single fusion protein thatretains the biological activity of both component polypeptides.

The fusion protein may additionally comprise a membrane-targeting domainso that the fusion protein is localised to the cell membrane. In oneembodiment, the polypeptide sequence additionally comprises amembrane-targeting domain.

The membrane-targeting domain may be a chemical modification orparticular protein sequence which attaches the molecule to the cellmembrane. Therefore, in one embodiment, the membrane-targeting region isselected from: a myristoylation-targeting sequence, apalmitoylation-targeting sequence, a prenylation sequence (i.e.,farnesylation, geranyl-geranylation, CAAX Box), a protein-proteininteraction motif or a transmembrane sequence (e.g. from a receptor).

In one embodiment, the fusion protein is a genetic fusion. In analternative embodiment, the fusion protein is generated using chemicalconjugation, for example using conventional chemical cross-linkers whichare used to fuse components A and B.

In one embodiment the fusion protein comprises the polypeptide sequenceand a ubiquitin targeting protein consisting of less than 135 aminoacids in length which comprises the hairpin motif of a cereblon bindingsite.In one embodiment the fusion protein comprises the polypeptide sequenceand a ubiquitin targeting protein, wherein the ubiquitin targetingprotein consists of a sequence selected from the group consisting of:SEQ ID NOs: 6-14 and 27.

Immunomodulatory Imide Drugs

In one embodiment, the compound is an immunomodulatory imide drug(IMiD). Such drugs are class of immunomodulatory drugs containing animide group. Currently, the primary use of IMiDs in the treatment ofcancers and autoimmune diseases.

In one embodiment, the IMiD is thalidomide, lenalidomide orpomalidomide, or a functional derivative or analog thereof. In a furtherembodiment, the IMiD is selected from the group consisting of:thalidomide, lenalidomide and pomalidomide or a functional derivative oranalog thereof.

Chimeric Antigen Receptors

According to a further aspect of the invention, there is provided achimeric antigen receptor (CAR) comprising:

-   -   an extracellular ligand binding domain;    -   a transmembrane domain;    -   an intracellular signalling domain; and,    -   a ubiquitin targeting protein as described herein, which is        capable of being bound by ubiquitin ligase in the presence of a        compound.

In one embodiment, the ubiquitination targeting domain consists of asequence selected from the group consisting of: SEQ ID NOs: 6-14 orfunctional fragments or functional variants thereof.

In one embodiment, the ubiquitination targeting domain consists of asequence selected from the group consisting of: SEQ ID NOs: 6-14, 27 orfunctional fragments or functional variants thereof.

As described herein, the compound (e.g. an IMiD) is capable of bindingto a ubiquitin ligase and enables the ubiquitin ligase to bind to theubiquitin targeting protein, thereby bringing the chimeric antigenreceptor into proximity of the ubiquitin ligase so that the chimericantigen receptor is capable of being ubiquitinated. Once ubiquitinated,the chimeric antigen receptor may be degraded by a proteasome or itssignalling activity may be impaired by the presence of the ubiquitinchain.

The CARs of the present invention include an intracellular ubiquitintargeting protein that may be bound by ubiquitin ligase in the presenceof a compound. By including a ubiquitin targeting protein in the CARconstruct, the CAR as expressed by the host cell can be readily andrapidly degraded upon exposure to a compound which mediates bindingbetween the ubiquitin targeting protein and ubiquitin ligase andutilizes the ubiquitin proteasomal pathway to degrade the CAR. In thisway, administering a compound targeting a ubiquitin targeting proteinwithin a CAR allows for the modulation of the activation of the CARexpressing cell, as degradation of the CAR or a portion thereof withinthe CAR expressing cell prohibits activation signalling from occurring.This strategy can be utilized to modulate the activation of the CARexpressing cell, for example, to lessen the activation of the CARexpressing cell in order to reduce adverse inflammatory responses.Furthermore, by utilizing this strategy, the host cell is spared andjust the CAR is degraded.

Furthermore, the use of the minimal degron as described herein, meanthat it is less likely to interfere with the signalling of the CARconstruct.

Standard chimeric antigen receptors are known in the art and generallycomprise a target binding domain, a transmembrane domain and anintracellular effector domain.

The target binding domain (also referred to as the extracellular ligandbinding domain) binds to a target, in particular wherein the target is atumour specific molecule, viral molecule, or any other moleculeexpressed on a target cell population that is suitable to mediaterecognition and elimination by a lymphocyte. In one embodiment, thetarget binding domain comprises an antibody, an antigen binding fragmentor a ligand. In one embodiment, the target binding domain comprises anantibody or fragment thereof. In one embodiment, the target bindingdomain is a ligand (e.g. a natural ligand of the target antigen). In analternative embodiment, the target binding domain is an antigen bindingfragment. In a further embodiment, the antigen binding fragment is asingle chain variable fragment (scFv) or a dAb. In a yet furtherembodiment, said scFv comprises the light (VL) and the heavy (VH)variable fragment of a target antigen specific monoclonal antibodyjoined by a flexible linker.

In one embodiment, the target binding domain may bind to more than onetarget, for example two different targets. Such a target binding domainmay be derived from a bispecific single chain antibody. For example,Blinatumomab (also known as AMG 103 or MT103) is a recombinant CD19 andCD3 bispecific scFv antibody consisting of four immunoglobulin variabledomains assembled into a single polypeptide chain. Two of the variabledomains form the binding site for CD19 which is a cell surface antigenexpressed on most normal and malignant B cells. The other two variabledomains form the binding site for CD3 which is part of the Tcell-receptor complex on T cells. These variable domains may be arrangedin the CAR in tandem, i.e. two single chain antibody variable fragments(scFv) tethered to a spacer, and transmembrane and signalling domains.The four variable domains can be arranged in any particular order withinthe CAR molecule (e.g. VL(first target)-VH(first target)-VH(secondtarget)-VL(second target) or VL(second target)-VH(secondtarget)-VH(first target)-VL(first target) etc.), joined with anysuitable linkers which are known in the art.

The target binding domain may bind a variety of cell surface antigens,but in one embodiment, the target binding domain binds to a tumourassociated antigen. In a further embodiment, the tumour associatedantigen is selected from: BCMA, carcinoembryonic antigen (CEA), cancerantigen-125, CA19-9, CD5, CD13, CD19, CD20, CD22, CD27, CD30, CD33,CD34, CD45, CD52, CD70, CD117, CD138, CD160, epidermal growth factorreceptor (EGFR), folate binding protein, ganglioside G2 (GD2), HER2,mesothelin, MUC-1, neural cell adhesion molecule (NCAM), prostate stemcell antigen (PSCA), prostate-specific membrane antigen (PSMA),prostatic acid phosphatise (PAP), protein melan-A, synaptophysis, sixtransmembrane epithelial antigen of the prostate I (STEAP1), TARP,Trp-p8, tyrosinase or vimentin. In a yet further embodiment, the tumourassociated antigen is BCMA.

In one embodiment the extracellular ligand binding domain is an anti-BCell maturation antigen (BCMA) single chain Fv amino acid sequence.

In one embodiment the extracellular ligand binding domain is ananti-BCMA single chain Fv amino acid sequence which comprises SEQ ID NO:29.

In one embodiment, the target binding domain has a binding affinity ofless than about 500 nanomolar (nM), such as less than about 400 nM, 350nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM,50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3nM, 2 nM, 1 nM, 0.5 nM or 0.25 nM. In one embodiment, the target bindingdomain has a binding affinity of about 10 nM to about 0.25 nM. In afurther embodiment, the target binding domain has a binding affinity ofabout 1 nM to about 0.5 nM (i.e. about 1000 pM to about 500 pM).

In one embodiment, the CAR additionally comprises a spacer domainbetween the target binding domain and the transmembrane domain. A spacerallows the target binding domain to orient in different directions tofacilitate binding and can be used to improve the target bindinginteraction. In one embodiment, the spacer comprises a sequence derivedfrom IgG (e.g. IgG1 Fc region or IgG1 hinge region), CD8 or CD4.

In one embodiment, the transmembrane domain can be derived either from anatural or from a synthetic source. In one embodiment, the transmembranedomain can be derived from any membrane-bound or transmembrane protein.Alternatively, the transmembrane domain can be synthetic and cancomprise predominantly hydrophobic residues such as leucine and valine.

For example, the transmembrane domain can be the transmembrane domain ofCD proteins, such as CD4, CD8, CD3 or CD28, a subunit of the T cellreceptor, such as α, β, γ or δ, a subunit of the IL-2 receptor (achain), or a subunit chain of Fc receptors. In one embodiment, thetransmembrane domain comprises the transmembrane domain of CD4, CD8 orCD28. In a further embodiment, the transmembrane domain comprises thetransmembrane domain of CD4 or CD8 (e.g. the CD8 alpha chain, asdescribed in NCBI Reference Sequence: NP_001139345.1, incorporatedherein by reference).

In one embodiment, the transmembrane domain comprises SEQ ID NO: 17.

The CAR may additionally comprise a hinge sequence next to thetransmembrane domain (e.g. between the target binding domain and thetransmembrane domain). Therefore, in one embodiment, the hinge sequencecomprises SEQ ID NO: 18. In a further embodiment, the hinge andtransmembrane domain comprise the complete sequence of SEQ ID NO: 19.

In some embodiments, the transmembrane domain is composed of the CD8atransmembrane helix immediately followed by the full lengthintracellular domain of 4-1BB which contains a stretch of sequencecompatible with the membrane interface. If the domain next to thetransmembrane domain does not have a sequence compatible with themembrane interface then a linker may be used.

Preferred examples of the intracellular effector domain for use in a CARdescribed herein, can be the cytoplasmic sequences of the natural T cellreceptor and co-receptors that act in concert to initiate signaltransduction following antigen binding, as well as any derivate orvariant of these sequences and any synthetic sequence that has the samefunctional capability. These domains can be separated into two classes:those that initiate antigen-dependent primary activation, and those thatact in an antigen-independent manner to provide a secondary orcostimulatory signal. Primary activation effector domains can comprisesignalling motifs which are known as immunoreceptor tyrosine-basedactivation motifs (ITAMs). ITAMs are well defined signalling motifs,commonly found in the intracytoplasmic tail of a variety of receptors,and serve as binding sites for syk/zap70 class tyrosine kinases.Examples of ITAMs used in the invention can include, as non-limitingexamples, those derived from CD3zeta, FcRgamma, FcRbeta, FcRepsilon,CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. Inone embodiment, the intracellular effector domain comprises a CD3zetasignalling domain (also known as CD247). In a further embodiment, theCD3zeta signalling domain comprises SEQ ID NO: 20. This sequence is alsofound in Uniprot P20963, residues 51-164. Natural TCRs contain a CD3zetasignalling molecule, therefore the use of this effector domain isclosest to the TCR construct which occurs in nature.

In one embodiment, the intracellular effector domain of the CARcomprises a CD3zeta signalling domain which has an amino acid sequencewith at least 70%, preferably at least 80%, more preferably at least85%, 90%, 95%, 97% or 99% sequence identity with SEQ ID NO: 20. In afurther embodiment, the intracellular effector domain of the CARcomprises a CD3zeta signalling domain which comprises an amino acidsequence of SEQ ID NO: 20.

The CAR may also provide a secondary or costimulatory signal. T cellsadditionally comprise costimulatory molecules which bind to cognatecostimulatory ligands on antigen presenting cells in order to enhancethe T cell response, for example by increasing proliferation activation,differentiation and the like. Therefore, in one embodiment, the CARadditionally comprises a costimulatory domain. In a further embodiment,the costimulatory domain comprises the intracellular domain of acostimulatory molecule, selected from CD28, CD27, 4-1BB (CD137), OX40(CD134), ICOS (CD278), CD30, CD40, PD-1 (CD279), CD2, CD7, NKG2C (CD94),B7-H3 (CD276) or any combination thereof. In a yet further embodiment,the costimulatory domain comprises the intracellular domain of acostimulatory molecule, selected from CD28, CD27, 4-1BB, OX40, ICOS orany combination thereof, in particular the intracellular domain of4-1BB.

In one embodiment, the costimulatory domain comprises a 4-1BB signallingdomain which has an amino acid sequence with at least 70%, preferably atleast 80%, more preferably at least 85%, 90%, 95%, 97% or 99% sequenceidentity with SEQ ID NO: 21. In a further embodiment, the costimulatorydomain comprises a 4-1BB signalling domain of SEQ ID NO: 21. Thissequence is also found in Uniprot Q07011, residues 214-255. Theadvantage of using this costimulatory domain is that it contains alysine residue which acts as a ubiquitination site (Lys219), thereforethe ubiquitin targeting protein used with a CAR construct containing the4-1BB costimulatory does not need to contain a ubiquitination siteitself in order for ubiquitination to be induced.

It will be understood that the intracellular components on the CAR (i.e.the signalling domain, costimulatory domain and ubiquitin targetingprotein) may be arranged in any order within the CAR construct, so longas they are located intracellularly. Therefore, in one embodiment theCAR construct comprises the domains in the following order:extracellular ligand binding domain—transmembrane domain—intracellularsignalling domain—ubiquitin targeting protein; extracellular ligandbinding domain—transmembrane domain—ubiquitin targetingprotein—intracellular signalling domain; extracellular ligand bindingdomain—transmembrane domain—intracellular signalling domain—ubiquitintargeting protein—costimulatory domain; extracellular ligand bindingdomain—transmembrane domain—costimulatory domain—intracellularsignalling domain—ubiquitin targeting protein; or extracellular ligandbinding domain—transmembrane domain—costimulatory domain—ubiquitintargeting protein—intracellular signalling domain. In a furtherembodiment, the CAR construct comprises the domains in the followingorder: extracellular ligand binding domain—transmembranedomain—costimulatory domain—ubiquitin targeting protein—intracellularsignalling domain.

In one embodiment the ubiquitin targeting protein is on the C-terminusof the CAR.

The nucleic acid sequence encoding the CAR may also compriseseparator/linker sequences between one or more of the domains of the CARconstruct. The linkers according to the invention may comprise alone, orin addition to other linkers, one or more sets of G, S or GS residues.In one embodiment, the linker comprises (GS)_(n) and/or (GGGGS)_(p)wherein n=1-10 and p=1-3. In one embodiment, the linker comprises GSGSGS(SEQ ID NO: 23), GSGSGSGSGS (SEQ ID NO: 24) or GGGGS (SEQ ID NO: 25).

According to a further aspect of the invention, there is provided amethod of controlling the activity of a chimeric antigen receptor celltherapy comprising:

(a) transducing or transfecting an immunomodulatory cell with apolynucleotide encoding the chimeric antigen receptor as describedherein;

(b) expressing said polynucleotide in the immunomodulatory cell;

(c) controlling the activation of the chimeric antigen receptor by theaddition of a compound;

wherein the compound mediates binding of a) the ubiquitin targetingprotein and b) a ubiquitin ligase in a manner that brings the chimericantigen receptor into proximity of the ubiquitin ligase, wherein thechimeric antigen receptor, in the presence of the compound, is capableof being ubiquitinated.

Without being bound by theory, addition of the compound is thought tolead to degradation of the CAR, thereby reducing (i.e. switching off)the CAR level and activity. By degrading at least a portion of the CAR,the ability of the CAR to activate the immune effector cell, for examplea CAR T-cell, is diminished. As contemplated herein, sufficientdegradation of the CAR occurs wherein the CAR's signalling functionalityis disrupted. Alternatively, the attachment of a polyubiquitin chaincould cause steric hindrance which leads to reduced activity throughsteric inhibition.

Signal Peptides

The components of the fusion protein described herein may comprise asignal peptide so that when a component is expressed in a cell, thenascent protein is directed to the endoplasmic reticulum andsubsequently to the cell surface where it may be expressed.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha helix. The signalpeptide may begin with a short positively charged stretch of amino acidswhich helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases. Thesignal peptide may be at the amino terminus of the molecule.

In one embodiment, the signal peptide is derived from CD8 (see UniProtP01732). In a further embodiment, the signal peptide comprises SEQ IDNO: 22 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations(insertions, deletions, substitutions or additions) provided that thesignal peptide still functions to cause cell surface expression of thecomponent (i.e. a functional variant).

Polynucleotides and Expression Vectors

According to a further aspect of the invention, there is provided anisolated polynucleotide encoding the ubiquitin targeting proteindescribed herein. According to a further aspect of the invention, thereis provided a polynucleotide encoding the fusion protein describedherein. According to a yet further aspect of the invention, there isprovided a polynucleotide encoding the chimeric antigen receptordescribed herein.

The polynucleotide sequences described herein may be codon optimised.The degeneracy found in the genetic code allows each amino acid to beencoded by between one and six synonymous codons allowing manyalternative nucleic acid sequences to encode the same protein(Gustafsson et al. (2004) Trends Biotechnol. 22(7): 346-53). Codonoptimisation is a technique used to modify genetic sequences with theintent of increasing the rate of expression of a gene in a heterologousexpression system; typically, the nucleotide sequence encoding a proteinof interest is codon optimized such that the codon usage more closelyresembles the codon bias of the host cell, while still coding for thesame amino acid sequence.

Nucleic acids described herein may comprise DNA or RNA. They may besingle-stranded or double-stranded. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification are well known in the art, such asmethylphosphonate and phosphorothioate backbones, or addition ofacridine or polylysine chains. Such modifications can be used in orderto enhance in vivo activity or life span of the polynucleotides of thepresent invention.

The polynucleotide may be present in an expression cassette orexpression vector (e.g. a plasmid for introduction into a bacterial hostcell, or a viral vector such as a lentivirus for transfection of amammalian host cell). Therefore, according to a further aspect of theinvention, there is provided an expression vector comprising any of thepolynucleotides described herein.

The term “vector” refers to a vehicle which is able to artificiallycarry foreign genetic material into another cell, where it can bereplicated and/or expressed. In one embodiment, the vector is a plasmid,a viral vector, a transposon based vector or a synthetic mRNA.

In one embodiment, the expression vector is a retroviral vector. In afurther embodiment, the retroviral vector is derived from, or selectedfrom, a lentivirus, alpha-retrovirus, gamma-retrovirus orfoamy-retrovirus, such as a lentivirus or gamma-retrovirus, inparticular a lentivirus. In a further embodiment, the retroviral vectorparticle is a lentivirus selected from the group consisting of HIV-1,HIV-2, SIV, FIV, EIAV and Visna. Lentiviruses are able to infectnon-dividing (i.e. quiescent) cells which makes them attractive vectorsfor gene therapy. In a yet further embodiment, the retroviral vectorparticle is HIV-1 or is derived from HIV-1. The genomic structure ofsome retroviruses may be found in the art. For example, details on HIV-1may be found from the NCBI Genbank (Genome Accession No. AF033819).HIV-1 is one of the best understood retroviruses and is therefore oftenused as a viral vector.

Host Cells

According to a further aspect of the invention, there is provided a cellcomprising the fusion protein described herein. According to anotheraspect of the invention, there is provided a cell comprising apolynucleotide or expression vector as described herein.

In one embodiment, the cell is an immunomodulatory cell. The term“immunomodulatory cell” refers to a cell of hematopoietic originfunctionally involved in the modulation (e.g. the initiation and/orexecution) of the innate and/or adaptive immune response. Saidimmunomodulatory cell according to the present invention can be derivedfrom a stem cell. The stem cells can be adult stem cells, non-humanembryonic stem cells, more particularly non-human stem cells, cord bloodstem cells, progenitor cells, bone marrow stem cells, inducedpluripotent stem cells, totipotent stem cells or hematopoietic stemcells. Said immunomodulatory cell can also be a dendritic cell, a killerdendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell. The T-cellmay be selected from the group consisting of inflammatory T-lymphocytes,cytotoxic T-lymphocytes, regulatory T-lymphocytes or helperT-lymphocytes, or a combination thereof. Therefore, in one embodiment,the immunomodulatory cell is derived from an inflammatory T-lymphocyte,cytotoxic T-lymphocyte, regulatory T-lymphocyte or helper T-lymphocyte.In another embodiment, said cell can be derived from the groupconsisting of CD4⁺ T-lymphocytes and CD8⁺ T-lymphocytes.

In one embodiment, the immunomodulatory cell may be a humanimmunomodulatory cell.

In one embodiment, the immunomodulatory cell is allogeneic orautologous. It will be understood that “autologous” refers to cellsobtained from the patient themselves, whereas “allogeneic” refers tocells obtained from a donor. Autologous cells have the advantage thatthey are compatible with the patient and therefore avoid anyimmunological compatibility problems leading to graft-versus-hostdisease (GvHD). In order to prevent the allogeneic cells from beingrejected by the patient, they would either need to be derived from acompatible donor or modified to ensure no antigens are present on thecell surface which would initiate an unwanted immune response.

Prior to expansion and genetic modification of the cells of theinvention, a source of cells can be obtained from a subject through avariety of non-limiting methods. Cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell can be derived from a healthy donor or a diseaseddonor, such as a patient diagnosed with cancer or an infection. Inanother embodiment, said cell is part of a mixed population of cellswhich present different phenotypic characteristics.

The immunomodulatory cells may be activated and/or expanded prior tobeing transduced with polynucleotides or expression vectors encoding thefusion protein described herein. For example, the cells may be treatedwith an anti-CD3 monoclonal antibody to cause activation.

It will be understood that the immunomodulatory cells may express thefusion protein described herein transiently or stably/permanently(depending on the transfection method used and whether thepolynucleotide encoding the fusion protein has integrated into theimmunomodulatory cell genome or not).

After introduction of the fusion protein, the immunomodulatory cells maybe purified.

Uses

The invention described herein provides for the use of a minimalubiquitin targeting protein as part of a safety switch. Therefore,according to an aspect of the invention there is provided the use of theubiquitin targeting protein described herein as a safety switch. In oneembodiment, the safety switch is used in a gene therapy (or methodthereof).

In a further embodiment, the gene therapy is a cellular gene therapy.

As described herein, the term “safety switch” refers to a biochemicalmechanism that can be activated on demand in order to control abiological process which can cause harm. Therefore, in one embodiment,the safety switch is used to control the signalling of a chimericantigen receptor (CAR) or heterologous T-cell receptor (TCR).

In one embodiment, the TCR is genetically modified. In a furtherembodiment, the T cell receptors affinity is changed to an affinityand/or specificity normally not present in said receptors naturalsurroundings. In a yet further embodiment, the T cell receptor'saffinity is changed to an affinity for a self-antigen, a tumour antigenand/or a pathogen derived antigen.

According to a further aspect of the invention, there is provided thecell described herein for use in therapy. In one embodiment, therapycomprises administration of the cell to a human subject in need of suchtherapy.

According to a further aspect of the invention, there is provided theuse of the fusion protein described herein, in a method of gene therapy.

In one embodiment, the therapy is adoptive cellular therapy. “Adoptivecellular therapy” (or “adoptive immunotherapy”) refers to the adoptivetransfer of human T lymphocytes that are engineered by gene transfer toexpress CARs or TCRs specific for surface molecules expressed on targetcells. This can be used to treat a range of diseases depending upon thetarget chosen, e.g. tumour specific antigens to treat cancer. Adoptivecellular therapy involves removing a portion of the patient's whiteblood cells using a process called leukapheresis. The T cells may thenbe expanded and mixed with expression vectors described herein in orderto permanently transfer the fusion protein to the T cells. The T cellsare expanded again and at the end of the expansion, the T cells arewashed, concentrated, and then frozen to allow time for testing,shipping and storage until the patient is ready to receive the infusionof engineered T cells.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a plurality of cells as definedherein. In one embodiment the cells comprise a polynucleotide sequenceencoding the polypeptide sequence and a ubiquitin targeting proteinconsisting of less than 135 amino acids in length which comprises thehairpin motif of a cereblon binding site. In one embodiment the cellscomprise an expression vector encoding the polypeptide sequence and aubiquitin targeting protein consisting of less than 135 amino acids inlength which comprises the hairpin motif of a cereblon binding site. Inone embodiment the cells comprising a polynucleotide sequence orexpression vector encoding the polypeptide sequence and a ubiquitintargeting protein consisting of less than 135 amino acids in lengthwhich comprises the hairpin motif of a cereblon binding site areimmunomodulatory cells. In one embodiment the cells comprising apolynucleotide sequence or expression vector encoding the polypeptidesequence and a ubiquitin targeting protein consisting of less than 135amino acids in length which comprises the hairpin motif of a cereblonbinding site are T-cells.

Examples of additional pharmaceutical composition ingredients include,without limitation, any adjuvants, carriers, excipients, glidants,sweetening agents, diluents, preservatives, dyes/colourants, flavourenhancers, surfactants, wetting agents, dispersing agents, suspendingagents, stabilizers, isotonic agents, solvents, surfactants,emulsifiers, buffers (such as phosphate buffered saline (PBS)),carbohydrates (such as glucose, mannose, sucrose or dextrans), aminoacids, antioxidants or chelating agents (such as EDTA or glutathione).

In one embodiment, the pharmaceutical composition additionally comprisesa pharmaceutically acceptable excipient, carrier, or diluent. Thecarrier, excipient or diluent must be “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notdeleterious to the recipient thereof. According to the present inventionany excipient, vehicle, diluents or additive used would have to becompatible with the fusion protein described herein. Standard textsknown in the art, such as “Remington's Pharmaceutical Science”, 17thEdition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations.

Pharmaceutical compositions may be administered by injection orcontinuous infusion (examples include, but are not limited to,intravenous, intratumoural, intraperitoneal, intradermal, subcutaneous,intramuscular and intraportal). In one embodiment, the composition issuitable for intravenous administration. When administering atherapeutic composition of the present invention (e.g., a pharmaceuticalcomposition containing a genetically modified cell as described herein),it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). Pharmaceutical compositions may besuitable for topical administration (which includes, but is not limitedto, epicutaneous, inhaled, intranasal or ocular administration) orenteral administration (which includes, but is not limited to, oral orrectal administration).

Methods for the preparation of such pharmaceutical compositions are wellknown to those skilled in the art. Other excipients may be added to thecomposition as appropriate for the mode of administration and theparticular protein used.

Effective doses and treatment regimens for administering the compositionof the present invention may be dependent on factors such as the age,weight and health status of the patient and disease to be treated. Suchfactors are within the purview of the attending physician.

According to a further aspect of the invention, there is provided apharmaceutical composition as defined herein, for use in the treatmentor prevention of a disease.

In one embodiment, the disease is selected from: a cancer, a pathogenicimmune response and an infection.

According to a further aspect of the invention, there is provided theuse of a pharmaceutical composition as described herein, in themanufacture of a medicament for the treatment and/or prevention of adisease.

Kits

According to a further aspect of the invention, there is provided a kitwhich comprises the fusion protein, chimeric antigen receptor,polynucleotide, expression vector, cell and/or pharmaceuticalcomposition as described herein.

Methods

According to a further aspect of the invention, there is provided amethod of engineering an immunomodulatory cell (i.e. to express thefusion protein described herein), comprising:

(a) providing an immunomodulatory cell;

(b) transducing or transfecting the polynucleotide or the expressionvector as defined herein, into said immunomodulatory cell; and

(c) expressing said polynucleotide or said expression vector in theimmunomodulatory cell.

In one embodiment, the immunomodulatory cell is obtained from a sampleisolated from a patient (i.e. autologous). In an alternative embodiment,the immunomodulatory cell is obtained from a donor (i.e. allogeneic).

As a non-limiting example, the fusion protein can be introduced as atransgene encoded by an expression vector as described herein. Theexpression vector can also contain a selection marker which provides foridentification and/or selection of cells which received said vector.

Polypeptides may be synthesized in situ in the cell as a result of theintroduction of polynucleotides encoding said fusion protein into thecell. Alternatively, said polypeptides could be produced outside thecell and then introduced thereto. Methods for introducing apolynucleotide construct into cells are known in the art and including,as non-limiting examples, stable transformation methods wherein thepolynucleotide construct is integrated into the genome of the cell ortransient transformation methods wherein the polynucleotide construct isnot integrated into the genome of the cell and virus mediated methods.Said polynucleotides may be introduced into a cell by, for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomesand the like. For example, transient transformation methods include forexample microinjection, electroporation or particle bombardment. Thepolynucleotides may be included in vectors, more particularly plasmidsor viruses, in view of being expressed in cells.

The terms “transfection”, “transformation” and “transduction” as usedherein, may be used to describe the insertion of the expression vectorinto the target cell. Insertion of a vector is usually calledtransformation for bacterial cells and transfection for eukaryoticcells, although insertion of a viral vector may also be calledtransduction. The skilled person will also be aware of the differentnon-viral transfection methods commonly used, which include, but are notlimited to, the use of physical methods (e.g. electroporation, cellsqueezing, sonoporation, optical transfection, protoplast fusion,impalefection, magnetofection, gene gun or particle bombardment),chemical reagents (e.g. calcium phosphate, highly branched organiccompounds or cationic polymers) or cationic lipids (e.g. lipofection).Many transfection methods require the contact of solutions of plasmidDNA to the cells, which are then grown and selected for a marker geneexpression.

Once the fusion protein has been introduced into the immunomodulatorycell, said cell may be referred to as a “transduced cell”. Therefore,according to a further aspect of the invention, there is provided a cellobtained by the method described herein. Also within the scope of thepresent invention is a cell line obtained from a transduced cellaccording to the method described herein.

According to a further aspect of the invention, there is provided amethod of inhibiting a CAR system in a subject which comprises theimmunomodulatory cells defined herein, which comprises administering tothe subject a compound which mediates binding between the ubiquitintargeting protein and a ubiquitin ligase. Such a compound would bringthe CAR into proximity of the ubiquitin ligase so that the CAR iscapable of being ubiquitinated. The ubiquitinated CAR can then degradedby a proteasome.

The level of CAR signalling by the system described herein, may beadjusted by altering the amount of compound present, or the amount oftime the compound is present. Therefore, in one embodiment, the level ofCAR cell activation may be increased by decreasing the dose of compoundadministered to the subject or decreasing the frequency of itsadministration. In an alternative embodiment, the level of CAR cellactivation may be reduced by increasing the dose of the compound, or thefrequency of administration to the subject.

Without being bound by theory, higher levels of CAR signalling arelikely to be associated with reduced disease progression but potentiallyincreased toxic activities, whilst lower levels of CAR signalling arelikely to be associated with increased disease progression butpotentially reduced toxic activities.

According to a further aspect of the invention, there is provided amethod of treating and/or preventing a disease, which comprisesadministering to a subject the cell or the pharmaceutical composition asdefined herein.

In one embodiment, the disease is cancer. In a further embodiment, thecancer is selected from: blood, bone marrow, lymph, lymphatic system,bladder, breast, colon, cervix, esophagus, kidney, large intestine,lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach.In a yet further embodiment, the cancer is a blood cancer, for exampleselected from the group consisting of: B cell leukaemia, multiplemyeloma (MM), acute lymphoblastic leukaemia (ALL), chronic lymphocyticleukaemia (CLL) and non-Hodgkin's lymphoma.

When the method described herein is used to treat cancer, in oneembodiment, the method reduces the number of tumour cells, reduces thetumour size and/or eradicates the tumour in the subject.

In one embodiment, the disease is a pathogenic immune response, such asan autoimmune disease, allergy or graft-versus-host rejection.Autoimmune diseases arise from an abnormal immune response of the bodyagainst substances and tissues normally present in the body. This canresult in the damage or destruction of tissues, or altered organ growthor function. Examples of autoimmune diseases include, but are notlimited to: diabetes mellitus Type 1, arthritis (including juvenile,psoriatic, reactive, and rheumatoid arthritis), psoriasis, multiplesclerosis, vasculitis, alopecia areata, pernicious anaemia,glomerulonephritis, autoimmune hepatitis, autoimmune pancreatitis,ulcerative colitis, systemic lupus erythematosus, Graves' disease,Guillain-Barré syndrome, Sjogren's syndrome, Celiac disease, Crohn'sdisease and Wegener's syndrome.

In one embodiment, the disease is an infection. An infection can becaused by a pathogen, such as a bacteria, virus, parasite, protozoa orfungi. In a further embodiment, the infection is a viral or bacterialinfection.

In one embodiment, the subject is a mammal. In a further embodiment, themammal is selected from the group consisting of: a human, a mouse, aprimate, a cow, a pig, a horse, a sheep, a cat, and a dog. In a yetfurther embodiment, the subject is a human.

The method of treatment and/or prevention, may comprise the followingsteps:

(a) providing a cell(s);

(b) transducing or transfecting the polynucleotide or the expressionvector as defined herein, into said cell(s);

(c) expressing said polynucleotide or said expression vector in thecell(s); and

(d) administering the cell(s) to a patient.

In one embodiment, the method additionally comprises: (e) administeringa compound which mediates binding between the ubiquitin targetingprotein and a ubiquitin ligase. This can be used to control the leveland/or activity of the polypeptide sequence expressed by saidpolynucleotide or said expression vector. The compound may beadministered to the patient before or simultaneously with thepolynucleotide or the expression vector (i.e. prior to or during step(d) in the method of treatment steps outlined above). In the context ofa CAR as described herein, administration of the compoundbefore/simultaneously with the polypeptide/expression vector allows theCAR to be administered in an “inactive” or a “lowly active” (i.e. OFF)state. The amount of agent can then be decreased in order to activatethe CAR. Administering the CAR in its inactive state allows for an evendistribution of the immunomodulatory cells to be achieved, thereforepreventing local accumulation of activated cells.

Alternatively, the compound may be administered to the patient afteradministration of the polynucleotide or the expression vector (i.e.after step (d) in the method of treatment steps outlined above) so thatthe CAR is administered in its “active” (i.e. ON) state.

The cells or pharmaceutical compositions described herein may beadministered to a patient who already has the disease in order tolessen, reduce or improve at least one symptom associated with thedisease and/or to slow down, reduce or block the progression of thedisease (i.e. therapeutically). The cells or pharmaceutical compositionsdescribed herein may be administered to a patient who has not yetcontracted the disease and/or who is not showing any symptoms of thedisease to prevent the cause of the disease (i.e. prophylactically). Thepatient may have a predisposition for, or be thought to be at risk ofdeveloping the disease.

The compound may be administered in the form of a pharmaceuticalcomposition. In this embodiment, the composition may additionallycomprise pharmaceutically acceptable carriers, diluents or excipients asoutlined herein.

The present invention provides a suitable OFF switch to be used withCAR-T cell therapies. The method may involve monitoring toxic activityin the patient. Thus, if the level of toxic activity becomes too high,the method can involve administering a compound enables ubiquitin ligaseto bind to the ubiquitin targeting protein and thus ubiquitinate thepolypeptide sequence/CAR, in order to reduce adverse toxic side effects.Toxic activities include, for example, immunological toxicity, biliarytoxicity and respiratory distress syndrome.

Similarly, the method may involve monitoring the progression of diseaseand then administering a compound which mediates binding between theubiquitin targeting protein and a ubiquitin ligase, and thusubiquitinate the polypeptide sequence/CAR, when an acceptable level ofdisease progression is reached (e.g. amelioration). The specific levelof disease progression determined to be “acceptable” will vary accordingto the specific circumstances and should be assessed on such a basis.

Monitoring the progression of the disease means to assess the symptomsassociated with the disease over time to determine if they arereducing/improving or increasing/worsening.

According to a further aspect of the invention, there is provided acompound for inhibiting the CAR as defined herein.

The invention may be described in more detail with reference to thefollowing, non-limiting examples:

Example 1: Design of Chimeric Antigen Receptors that IncorporateCereblon Binding Motifs as a Strategy to Build a Molecular Off-Switch

Constructs have been designed to build a chimeric antigen receptoroff-switch using the cereblon binding motifs present in human cereblontargets such as Ikaros3, Casein kinase 1 alpha and GSTP1. Suchconstructs aim to 1) be degraded by the addition of small molecules suchas lenalidomide b) signal as chimeric antigen receptors by theactivation of the NFAT pathway in the absence of compound and c) beturned off by the addition of compounds.

Experiments have been designed to measure the degradation of GFPproteins containing Ikaros3 ZnF cereblon binding regions and theobserved structural conservation on the cereblon binding sites of threeprotein substrates Ikaros3 ZNF2, CK1 alpha and GSTP1:

TABLE 3 Cereblon binding sites Substrate UniProt ID Structural reference(PDB entries) Ikaros3 Q9UKT9 PDB entries 2MA7 and 2I13 will be used asZNF2 structural representatives of Ikaros3 ZnF: 2MA7 with sequenceidentity of 95% when aligned to Ikaros 3 residues 131-175 (SEQ ID NO: 8)and 2I13 with sequence identity of 53% when aligned to Ikaros 3 residues131-175 (SEQ ID NO: 8). 2I13 will be used as a model of Ikaros3as thestructure is represented by a single experimental model. GSTP1 P151705HXB CK1 alpha P48729 5FQD

Structural conservation of the cereblon binding sites in the threesubstrates: Ikaros3, casein kinase I and GSTP1, was observed. However,this was in the absence of sequence conservation (see SEQ ID NOs: 2-4).When the three hairpin motifs from the substrates are superposed usingmain chain atoms the overall rmsd is around 1.7 Å:

TABLE 4 Pairwise RMSD Matrix Chains GSTP1 CK1 alpha Ikaros3 (from 2I13)GSTP1 2.04 1.81 CK1 alpha 2.04 1.38 Ikaros3 (from 2I13) 1.81 1.38

The closest structural overlap is seen around a central Glycine residueand the rmsd starts to increase as the residues are located farther awayfrom the central glycine, in particular on the C-terminal direction:

TABLE 5 Individual residue RMSD GSTP1 vs CK1 GSTP1 vs Ikaros3 CK1 vsIkaros3 Pair rmsd Pair rmsd Pair rmsd ILE 35 - VAL 0.758 LYS 51 - VAL0.595 LYS 51 - ILE 0.348 570: 570: 35: ASN 36 - ASP 0.629 CYS 52 - ASP0.65 CYS 52 - ASN 0.386 571: 571: 36: ILE 37 - LYS 0.166 PRO 53 - LYS0.608 PRO 53 - ILE 0.556 572: 572: 37: THR 38 - LYS 0.22 GLU 54 - LYS0.328 GLU 54 - THR 0.443 573: 573: 38: ASN 39 - SER 0.435 CYS 55 - SER0.429 CYS 55 - ASN 0.37 574: 574: 39: GLY 40- GLY 0.208 GLY 56 - GLY0.63 GLY 56 - GLY 0.426 575: 575: 40: GLU 41 - GLU 0.245 LYS 57 - GLU0.562 LYS 57 - GLU 0.39 576: 576: 41: GLU 42 - LYS 0.477 SER 58 - LYS0.431 SER 58 - GLU 0.051 577: 577: 42: VAL 43 - SER 0.733 PHE 59 - SER0.974 PHE 59-VAL 0.947 578: 578: 43: ALA 44 - LYS 4.683 SER 60 - LYS4.139 SER 60-ALA 2.335 579: 579: 44: ALA 44 - LYS 4.683 ASP 61 - THR3.15 ASP 61-VAL 2.624 579: 580: 45: LYS 46 - ARG 5.507 LYS 62 - ARG 6.03LYS 62-LYS 4.847 581: 581: 46:

An alternative representation of the structural similarity is theRamachandran angles as they describe the conformation of the peptidemain-chain. When Ramachandran angles are measured in these cereblonbinding motifs we have (central glycine is highlighted in bold):

TABLE 6 Ramachandran angles of cereblon binding hairpin motifs CK1 GSTP1Ikaros3 (2MA7) Residue Psi Phi Residue Psi Phi Residue Psi Phi ILE 35129.7 −125.5 VAL 570 132.4 −149.2 LYS 51 151.5 −102.8 ASN 36 106.9 −70.6ASP 571 141 −82.1 CYS 52 127.8 −83.5 ILE 37 −14.2 −60.3 LYS 572 −42.5−79.4 PRO 53 −7.5 −66 THR 38 −18.3 −89.2 LYS 573 −47.1 −52.6 GLU 54−52.5 −111.7 ASN 39 −3 −152 SER 574 −65.9 −90.8 CYS 55 −16.9 −105.2 GLY40 −4.3 91.8 GLY 575 −4.8 123.6 GLY 56 −4.9 91.2 GLU 41 131.7 −56.5 GLU576 104.1 −75.1 LYS 57 127.9 −60.6 GLU 42 133.3 −96.6 LYS 577 169 −52.5SER 58 148.5 −97.5 VAL 43 169.4 −136.4 SER 578 168.1 −108.3 PHE 59 155.5−131.2 ALA 44 139.7 −106.3 LYS 579 −59.1 −91.6 SER 60 −32.1 −73.3 VAL 45112 −127.1 THR 580 169.3 −86.4 ASP 61 161.1 −132.6 LYS 46 125.4 −87.9ARG 581 156.7 −64.6 LYS 62 −35.3 −69

Direct comparison of the differences can be facilitated by calculationof the modulus of the differences in the angles, so if the difference is0 the conformation is identical and the higher positive values thehigher is the overall difference in conformation. The pairwisecomparison of the Ramachandran angles differences (delta) represented asthe modulus of the angle difference (angle 1-angle 2) is shown in Table7:

TABLE 7 Comparison between Ramachandran angles CK1 vs GSTP1 CK1 vsIkaros GSTP1 vs Ikaros delta delta delta delta delta delta Residue PsiPhi Psi Phi Psi Phi −5 2.7 23.7 21.8 22.7 19.1 46.4 −4 34.1 11.5 20.912.9 13.2 1.4 −3 28.3 19.1 6.7 5.7 35 13.4 −2 28.8 36.6 34.2 22.5 5.459.1 −1 62.9 61.2 13.9 46.8 49 14.4 1 (Gly) 0.5 31.8 0.6 0.6 0.1 32.4 227.6 18.6 3.8 4.1 23.8 14.5 3 35.7 44.1 15.2 0.9 20.5 45 4 1.3 28.1 13.95.2 12.6 22.9 5 198.8 14.7 171.8 33 27 18.3 6 57.3 40.7 49.1 5.5 8.246.2 7 31.3 23.3 160.7 18.9 192 4.4

Residue 1 is the central Glycine residue. In this comparison, it isclear the conformational similarity of CK1 and Ikaros3 around residues 1to 4 with Psi angles differences <20° and Phi angles differences <5°.Comparison of both CK1 and Ikaros3 with GSTP1 in this region shows thesame conformational trend but with higher variations of the angles withPsi variations <35° and Phi variations <45°. In all cases, thestructural differences become apparent around position 5 and this is inagreement with the rmsd differences.

Based on data available and structural analysis it is proposed that the2nd zinc finger of Ikaros3 should provide a “minimal” degron in the formof a small protein domain (around 30 amino acids) that can be added to aprotein of interest for degradation and in the presence of animmunomodulatory imide drug (IMiD) degradation will be induced. Thisdegron/ubiquitin targeting protein can therefore be incorporated intoCAR architecture using methods known in the art in order to create a CARconstruct with an “off switch”.

Example 2. Proximity Induced Degradation of Green Fluorescent Protein(GFP) Protein Fused to Ikaros 1 Derived Degron Sequences in HeLa Cells

This example illustrates the selective degradation of GFP in thepresence of lenalidomide. GFP constructs are composed of GFP codingsequence in frame with a (Glycine-Serine)×N linker of lengths N=1, 3, 5followed by a degron sequence derived from human Ikaros 1. Experimentswere conducted with constructs transfected in HeLa cells and degradationwas followed by flow cytometry.

Materials and Methods Generation of Constructs:

GFP constructs were cloned into pTT5 vector (FIG. 2) fused to a(Glycine-Serine)×N linker of length N=1, 3, 5 and human Ikaros 1 (IKFZ1,Uniprot Q13422) sequence containing residues 141 to 168 (SEQ ID NO:27).Full sequence details of the constructs are given below.

CONSTURCT 1 (SEQ ID NO 30)MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYL

Legend: GFP Linker 4 (SEQ ID NO 26)

CONSTRUCT 1 DNA sequence (SEQ ID NO 31)ATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTTCAGGAGAACGGCCCTTCCAGTGCAATCAGTGCGGGGCCTCATTCACCCAGAAGGGCAACCTGCTCCGGCACATCAAGCTGCA TTCC CONSTRUCT 2(SEQ ID NO 32) MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY

Legend: GFP Linker 1 (SEQ ID NO 23)

CONSTUCT 2 DNA sequence (SEQ ID NO 33)ATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTTCAGGTTCAGGTTCAGGAGAACGGCCCTTCCAGTGCAATCAGTGCGGGGCCTCATTCACCCAGAAGGGCAACCTGCTCCGGCA CATCAAGCTGCATTCCCONSTRUCT 3 (SEQ ID NO 34)MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY

Legend: GFP Linker 2 (SEQ ID NO 24)

CONSTRUCT 3 DNA sequence (SEQ ID NO 35)ATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTTCAGGTTCAGGTTCAGGTTCAGGTTCAGGAGAACGGCCCTTCCAGTGCAATCAGTGCGGGGCCTCATTCACCCAGAAGGGCAACCTGCTCCGGCACATCAAGCTGCATTCCTransfection of HeLa Cells with CONSTRUCTS 1, 2 and 3 and LenalidomideTreatment:

HeLa Ohio cells grown in EMEM (EBSS)+2 mM Glutamine+1% Non-EssentialAmino Acids (NEAA)+10% Foetal Bovine Serum (FBS) supplemented with 10%heat inactivated foetal bovine serum (Gibco) and 50 U/mL penicillin+50μg/mL streptomycin (Gibco) were transfected with 0.5 μg of constructplasmid using Lipofectamine 2000 reagent (Thermofisher). Aftertransfection, cells were incubated at 37° C. in a CO₂ incubator for 24hours. lenalidomide was reconstituted to 10 mM in 100% DMSO and dilutedto 1 mM, 0.5 mM, 0.1 mM and 0.05 mM in 100% DMSO. lenalidomide was addedto the cell medium to a final concentration of 10 μM, 1 μM, 0.5 μM, 0.1μM, and 0.01 μM and the corresponding DMSO amount for the no compoundcontrol. The final DMSO concentration in all conditions was 0.1%. Afterlenalidomide treatment, cells were incubated at 37° C., 5% CO₂ for 24hours. GFP expression was measured by flow cytometry with an iQue(Intellicyt) and data analysed using ForeCyt (Intellicyt).

Results

Lenalidomide induced degradation of GFP encoded in CONSTRUCTS 1, 2 and 3was assessed by measurement of the median fluorescence intensity (MFI)of GFP positive cells.

The effect of lenalidomide on the expression level of GFP is displayedon FIG. 3.

Example 3: Proximity Induced Degradation of Chimeric Antigen Receptor(CAR) Constructs Transfected in Jurkat Cells

This example illustrates the selective degradation of CAR constructs inthe presence of lenalidomide. Experiments were conducted by transfectionof the CAR constructs in Jurkat cells.

Materials and Methods Generation of Constructs:

Two constructs were generated to evaluate the effect of lenalidomide onthe regulation of the expression levels of a receptor (CAR). CONSTRUCT 4is a conventional CAR with an antigen recognition scFv that binds to andis activated by the B-cell maturation antigen BCMA (Uniprot Q02223). ThescFv is followed by human CD8a hinge and transmembrane domain, human4-1BB co-stimulatory and the human CD3 intracellular domains (FIG. 1).CONSTRUCT 5 contains the same elements of CONSTRUCT 4 plus theC-terminal end addition of two sections of the human Ikaros 3 protein(Uniprot Q9UKT9). The first section comprises residues 131-175 (SEQ IDNO:27) and is followed by a section containing residues 231-249 (SEQ IDNO: 28). Full sequence details of the constructs are given below.

CONSTRUCT 4 (SEQ ID NO 36) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEWIGATYRGHSDTYYNQKFKGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

Legend: CD8α (Uniprot P01732) signal peptide (SEQ ID NO 22)Anti-BCMA single chain Fv (SEQ ID NO 29)

4-1BB costimulatory domain (SEQ ID NO 21)

CONSTRUCT 4 DNA Sequence (SEQ ID NO 37)ATGGCCCTGCCCGTGACCGCCCTCCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGGCCCCAGGTCCAGCTGGTGCAGAGCGGGGCCGAGGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAGCTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGGACAGGGACTGGAGTGGATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAAGTTCAAGGGCAGGGCCACCCTGACCGCCGACACTAGCACCAGCACCGCCTACATGGAACTGAGCTCACTGCGGAGCGAGGACACCGCCGTGTACTACTGCACCAGGGGCGCCATCTACGACGGCTACGACGTGCTGGACAACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCTGGCGGCGGCGGGAGCGGCGGCGGCGGAAGCGGCGGCGGAGGAAGCGGCGGCGGCGGAAGCGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGCGCAAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCTCTAACCTGCACAGCGGCGTGCCCAGCAGGTTCTCTGGCAGCGGCTCCGGCACCGACTTCACTCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGGAAGCTCCCCTGGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGCTTCGTGCCCGTGTTCCTCCCCGCAAAACCCACCACCACTCCCGCCCCCAGACCCCCCACTCCCGCCCCAACAATTGCCAGCCAGCCCCTGAGCCTGAGGCCCGAGGCTTGTAGGCCCGCCGCTGGCGGCGCCGTCCACACCAGGGGCCTGGACTTCGCCTGCGACATCTATATCTGGGCCCCCCTGGCCGGAACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCACAGGAACAAGAGGGGCAGGAAGAAGCTCCTGTACATCTTCAAGCAGCCCTTCATGAGGCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCAGGTTCCCAGAGGAAGAGGAGGGCGGGTGCGAACTGAGAGTGAAATTTAGCAGGAGCGCCGACGCCCCCGCCTATCAGCAAGGCCAGAACCAGCTGTACAACGAGCTCAACCTGGGCAGGAGGGAGGAGTACGACGTGCTGGACAAGCGGAGGGGCAGAGATCCCGAGATGGGCGGCAAGCCCAGGAGGAAGAATCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGGGAGAGGAGGAGGGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCAC ATGCAGGCCCTGCCCCCCAGGCONSTUCT 5 (SEQ ID NO 38) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEWIGATYRGHSDTYYNQKFKGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

Legend: CD8α (Uniprot P01732) signal peptide (SEQ ID NO 22)Anti-BCMA single chain Fv (SEQ ID NO 29)

4-1BB costimulatory domain (SEQ ID NO 21)

Ikaros 3 residues 131-175 (Uniprot Q9UKT9) (SEQ ID NO 8)Ikaros 3 residues 231-249 (Uniprot Q9UKT9) (SEQ ID NO 28)CONSTRUCT 5 DNA sequence (SEQ ID NO 39)ATGGCTCTTCCTGTAACCGCACTTCTGCTTCCTCTTGCTCTGCTGCTTCATGCTGCTAGACCTCAGGTGCAGTTAGTGCAATCTGGAGCTGAGGTGAAGAAACCTGGCTCTTCCGTGAAAGTGAGCTGTAAGGGAAGCGGCTACACCTTTACCAACTACTGGATGCATTGGGTGAGACAGGCCCCTGGACAGGGATTAGAGTGGATTGGAGCCACATATAGAGGACACAGCGATACCTACTACAACCAGAAGTTCAAGGGCAGGGCCACCCTTACAGCCGATACAAGCACATCTACCGCCTACATGGAACTGTCTTCTCTGAGAAGCGAGGATACCGCCGTGTACTACTGCACAAGAGGAGCCATCTACGACGGCTATGATGTTCTGGACAATTGGGGACAGGGCACACTGGTGACAGTGTCTTCTGGTGGTGGCGGGTCCGGTGGAGGCGGATCTGGCGGTGGGGGCTCCGGAGGAGGAGGTTCAGATATTCAAATGACACAGAGCCCAAGCAGCCTGTCTGCTTCTGTGGGCGATAGAGTGACCATCACCTGTTCTGCTTCTCAGGATATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAAGCCCCTAAACTGCTGATCTACTACACCAGCAATCTGCACTCTGGAGTTCCTAGCAGATTCAGCGGAAGCGGCTCTGGCACCGATTTTACACTGACCATCTCTTCTCTGCAGCCTGAGGATTTTGCCACCTACTACTGCCAGCAGTACCGGAAATTGCCTTGGACCTTTGGACAGGGAACCAAGCTGGAGATCAAGAGGTTTGTGCCCGTGTTTCTGCCTGCTAAGCCTACAACAACACCTGCCCCTAGACCACCTACACCTGCTCCTACAATTGCCTCTCAGCCTCTTTCTCTGAGACCTGAAGCTTGCAGACCTGCTGCTGGAGGAGCTGTGCATACAAGAGGACTGGATTTTGCCTGCGATATCTACATTTGGGCTCCACTGGCCGGCACATGTGGAGTTCTTCTGCTGTCTCTGGTGATCACCCTGTACTGTAATCACAGGAACAAGCGGGGCCGGAAAAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCAGTTCAGACAACACAGGAGGAGGACGGCTGTAGCTGCAGATTTCCTGAGGAAGAGGAAGGAGGATGTGAATTAGGTGGTGGCGGGAGCAGGGTGAAGTTCTCACGCAGCGCAGATGCTCCTGCCTATCAGCAAGGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGATGTGCTGGACAAGAGAAGGGGCAGAGATCCTGAAATGGGAGGAAAGCCCAGAAGGAAGAACCCTCAAGAAGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATTGGCATGAAAGGAGAGAGGAGAAGAGGAAAGGGCCATGATGGCCTGTATCAGGGCCTGTCTACAGCCACCAAGGATACATATGATGCCCTGCATATGCAGGCTTTACCCCCTAGAGGAGGAGGCGGATCTAACGTGCTGATGGTGCATAAAAGAAGCCACACAGGAGAGAGACCATTCCAGTGCAACCAGTGTGGAGCCAGCTTCACCCAGAAGGGAAATCTGCTGAGACACATCAAACTGCATACAGGCGAGAAGCCCTTCAAGGACCCTGGCGATACAGCCTCTGCTGAAGCTAGACACATTAAAGCCGAAATGGGC

Expression of Constructs in Jurkat Cells:

NFAT-luc2 Jurkat cells (Promega) to a density of 2×10⁵ cells/ml werecultured in RPMI medium 1640 (1×) without L-glutamine with phenol red(Gibco), 10% (v/v) Fetal Bovine Serum (FBS) Heat-Inactivated (Gibco), 1%(v/v) Minimum essential medium non-essential amino acids (MEM NEAA)(ThermoFisher), 1% (v/v) Sodium Pyruvate (Sigma), 1% (v/v) L-Glutamine(Gibco). 20 μg of plasmid DNA was mixed with 8×10⁶ NFAT-luc2 Jurkatcells and cells were transfected using the 4D-Nucleofector (Lonza) withcell Line SE Nucleofector kit (Lonza) by following manufacturerinstructions with program CL-120. Cells were incubated at 37° C. with 5%CO₂ for 48 h. Lenalidomide at a stock concentration of 10 mM in 100%DMSO was diluted in Jurkat media to achieve a stock concentration of 250μM. Using the 250 μM stock, NFAT-luc2 Jurkat cells were incubated at afinal compound concentration of 10 μM or 0 μM (DMSO in media) for 24hour at 37° C. with 5% CO₂. Final DMSO concentration was 0.1% in allwells. Cells were then stained with AlexaFluor 647 conjugated BCMA-Fc tolabel the anti-BCMA CAR. Measurements were made using a Cytoflex S(Beckman Coulter) and data analysed using FlowJo.

Results

FIG. 5 displays the effect of lenalidomide treatment on the expressionlevel of CAR molecules on the surface of Jurkat cells. CONSTRUCT 4,which does not contain any degron sequence is unaffected by the compoundwhile CONSTRUCT 5 expression is reduced by the addition of 10 μMlenalidomide.

Example 4. Proximity Induced Degradation of Chimeric Antigen Receptor(CAR) Constructs in Primary T-Cells. Effect of Lenalidomide on CytokineRelease

This example demonstrates that CAR containing degron sequences arefunctional in primary T-cells and are degraded by the addition oflenalidomide.

Materials and Methods

For lentiviral vector production, 3.0×10⁷ LentiX 293T (HEK 293T) cellswere seeded in 20 mL DMEM (Gibco) and were incubated overnight at 37° C.with 5% CO₂. LentiX cells were transfected by mixing, for example, 21 μgof transfer vector containing the construct, 3.75 μg ViraSafe pRSV-Rev,5.25 μg ViraSafe pCMV-VSVG, 7.5 μg ViraSafe pCgp V-(gag-pol), 75 μgjetPRIME (Polyplus) and 1500 μg jetPRIME Buffer (Polyplus). After 2days, supernatants were clarified and virus was concentrated andpurified by ultracentrifugation on a 20% sucrose cushion using Ultrapuresucrose (ThermoFisher) in 50 ml Oak Ridge PPCO ultracentrifugation tubes(ThermoFisher). Lentiviral vectors were produced for CONSTRUCT 4 andCONSTRUCT 5 using the method described above.

Peripheral blood mononuclear cells (PBMCs) from the fresh blood of threehealthy human donors were isolated by density gradient centrifugation inAccuspin tubes (Sigma) containing 15 mL of Histopaque-1077 (Sigma) andfollowing manufacturer's instructions. Cells were resuspended at 1×10⁶cells/mL in TEXMacs media (Miltenyi Biotec) containing 100 units/mL ofIL-2 (Sigma) and TransAct beads (Miltenyi Biotec) and incubated for 48 hat 37° C. with 5% CO₂.

T-cells from the three donors were then transduced with the lentiviralvectors encoding for CONSTRUCT 4 and CONSTRUCT 5. Transduction reactionswere prepared to achieve an MOI of 5. T-cells were cultured in TEXMacsmedia with 100 units/mL of IL-2, fresh media was added every 3 days.ARH-77-10B5 cells, which express a high-level of the BCMA antigen, werecultured in Jurkat media (described in Example 3) plus 1 mg/mL G418(Gibco) at 37° C. with 5% CO₂.

7 days after transduction, lenalidomide at a stock concentration of 10mM in 100% DMSO was diluted in TEXMacs media to achieve a stockconcentration of 250 μM, and 5×10⁴ T-cells were incubated at a finalcompound concentration of 10 μM or 0 μM (DMSO in media) for 16 h at 37°C. with 5% CO₂. T-cells were then co-cultured (5×10⁵ cells per well, 1:1effector: target ratio) with either ARH-77-10B5 cells (BCMA positivecells) or media for 24 h in TEXMacs media containing either 10 μM or 0μM (DMSO in media) at 37° C. with 5% CO₂. Final DMSO concentration was0.1% in all wells. Cells were pelleted (1200 rpm, 5 min) andsupernatants were collected. Supernatants were analysed for cytokinelevels using MSD V-plex Proinflammatory Panel 1 Human Kit (MSD) and MSDSector Imager (MSD).

Results

Effect of lenalidomide treatment on the expression levels of CONSTRUCTS4 and 5 in transduced primary T-cells is shown in FIG. 6. After antigenpresentation, T-cell supernatants were analysed for their TNFα, IL2 andIFN-γ levels. Supernatants corresponding to CONSTRUCT 4, a CAR constructwithout degron domain, showed increased levels of cytokinesconcentrations when compared with no lenalidomide treatment in line withpublished data (Otahal P et al. 2016 Oncoimmunology Vol. 5, No. 4), FIG.7. This effect was also observed for cells transduced with CONSTRUCT 5(CAR plus degron elements) but at a reduced extent when compared withthe control CAR (CONSTRUCT 4), FIG. 7.

It will be understood that the embodiments described herein may beapplied to all aspects of the invention. Furthermore, all publications,including but not limited to patents and patent applications, cited inthis specification are herein incorporated by reference as though fullyset forth.

SEQUENCES Structure Sequence SEQ ID NO. Structural bindingX₁X₂X₃X₄X₅GX₇X₈X₉X₁₀ 1 motif consensus sequences Ikaros 3 (ZFN2) QCGASFT2 binding loop GSPT1 binding loop VDKKSGEKSK 3 CK1 alpha bindingINITNGEEVA 4 loop ZFP91 binding loop LQCEICGFTCR 5 Ikaros3 (141-173)TGERPFQCNQCGASFTQKGNLLRHIKLHSTGEKP 6 Ikaros3 (141-173)TGERPFQCNQCGASFTQKGNLLRHIKLHTGEKPS 7 plus Ub tag TDPGDTASAEARHIKAEMGIkaros (131-175) NVLMVHKRSHTGERPFQCNQCGASFTQKGNLLRH 8 (Uniprot Q9UKT9)IKLHTGEKPFK Ikaros3 (131-175) NVLMVHKRSHTGERPFQCNQCGASFTQKGNLLRH 9plus Ub tag (Ikaros 3 IKLHTGEKPFKDPGDTASAEARHIKAEMG residues 231-249(Uniprot Q9UKT9)) Ikaros3 (117-249) KMNCDVCGLSCISFNVLMVHKRSHTGERPFQCNQ10 CGASFTQKGNLLRHIKLHTGEKPFKCHLCNYACQ RRDALTGHLRTHSVEKPYKCEFCGRSYKQRSSLEEHKERCRTFLQSTDPGDTASAEARHIKAEMG GSTP1 (388-499)HSGRTFDAQIVIIEHKSIICPGYNAVLHIHTCIE 11 EVEITALICLVDKKSGEKSKTRPRFVKQDQVCIARLRTAGTICLETFKDFPQMGRFTLRDEGKTIAIG KVLKLVPEKD GSTP1 (388-499)HSGRTFDAQIVIIEHKSIICPGYNAVLHIHTCIE 12 F471S/M474SEVEITALICLVDKKSGEKSKTRPRFVKQDQVCIA RLRTAGTICLETFKDSPQSGRFTLRDEGKTIAIGKVLKLVPEKD CK1 alpha (8-94) KAEFIVGGKYKLVRKIGSGSFGDIYLAINITNGE 13EVAVKLESQKARHPQLLYESKLYKILQGGVGIPH IRWYGQEKDYNVLVMDLLG CK1 alpha (8-94)KAEFIVGGKYKLVKIGSGSFGDIYLAINITNGEE 14 L63H/L67Q/I73QVAVKLESQKARHPQLLYESKHYKIQQGGVGQPHI RWYGQEKDYNVLVMDLLGExpanded structural X₁X₂X₃X₄X₅GX₇X₈X₉X₁₀X₁₁X₁₂ 15 binding motifconsensus sequence Ubiquitination tag STDPGDTASAEARHIKAEMG 16 CD8αtransmembrane TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN 17 sequence HRN CD8αhinge sequence

FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVH 18 CD8α hinge andFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRP 19 transmembraneEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV sequence LLLSLVITLYCNHRN

CD3ζ domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV 20 (Uniprot P20963)LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4-1BB costimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE 21domain EEEGGCEL CD8α signal MALPVTALLLPLALLLHAARP 22 sequence(Uniprot P01732) Linker 1 GSGSGS 23 Linker 2 GSGSGSGSGS 24 Linker 3GGGGS 25 Linker 4 GS 26

GERPFQCNQCGASFTQKGNLLRHIKLHS 27 Ikaros 3 residues 231-DPGDTASAEARHIKAEMG 28 249 (Uniprot Q9UKT9) Anti-BCMA singleQVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWM 29 chain Fv Amino AcidHWVRQAPGQGLEWIGATYRGHSDTYYNQKFKGRA SequenceTLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIY (Uniprot Q02223)DGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR Construct 1 Amino MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 30Acid Sequence GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK GSGERPFQCNQCGASFTQKGNLLRHIKLHSContruct 1 DNA ATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG 31 SequenceTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAA CGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAG TCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC CGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTTCAGGAGAACGGCCCTTCCAGTGCAATCAGT GCGGGGCCTCATTCACCCAGAAGGGCAACCTGCTCCGGCACATCAAGCTGCATTCC Construct 2 AminoMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 32 Acid Sequence GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGV QCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSGSGSGERPFQCNQCGASFTQKGNLLRHIKLHS Construct 2 DNAATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG 33 SequenceTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAA CGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAG TCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC CGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTTCAGGTTCAGGTTCAGGAGAACGGCCCTTCC AGTGCAATCAGTGCGGGGCCTCATTCACCCAGAAGGGCAACCTGCTCCGGCACATCAAGCTGCATTCC Construct 3 AminoMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 34 Acid SequenceGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGV QCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSGSGSGSGSGERPFQCNQCGASFTQKGNLLRHI KLHS Construct 3 DNAATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG 35 SequenceTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAA CGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAG TCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC CGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTTCAGGTTCAGGTTCAGGTTCAGGTTCAGGAG AACGGCCCTTCCAGTGCAATCAGTGCGGGGCCTCATTCACCCAGAAGGGCAACCTGCTCCGGCACATC AAGCTGCATTCC Construct 4 AminoMALPVTALLLPLALLLHAARPQVQLVQSGAEVKK 36 Acid SequencePGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEW IGATYRGHSDTYYNQKFKGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGT LVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGK APKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRFV PVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPRConstruct 4 DNA ATGGCCCTGCCCGTGACCGCCCTCCTGCTGCCCC 37 SequenceTGGCCCTGCTGCTGCACGCCGCCAGGCCCCAGGT CCAGCTGGTGCAGAGCGGGGCCGAGGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAGCTGCAAGGGCA GCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGGACAGGGACTGGAGTGG ATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAAGTTCAAGGGCAGGGCCACCCT GACCGCCGACACTAGCACCAGCACCGCCTACATGGAACTGAGCTCACTGCGGAGCGAGGACACCGCCG TGTACTACTGCACCAGGGGCGCCATCTACGACGGCTACGACGTGCTGGACAACTGGGGCCAGGGCACC CTGGTGACAGTGAGCTCTGGCGGCGGCGGGAGCGGCGGCGGCGGAAGCGGCGGCGGAGGAAGCGGCGG CGGCGGAAGCGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGA CCATCACCTGCAGCGCAAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG GCCCCTAAGCTGCTGATCTACTACACCTCTAACCTGCACAGCGGCGTGCCCAGCAGGTTCTCTGGCAG CGGCTCCGGCACCGACTTCACTCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACT GCCAGCAGTACAGGAAGCTCCCCTGGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGCTTCGTG CCCGTGTTCCTCCCCGCAAAACCCACCACCACTCCCGCCCCCAGACCCCCCACTCCCGCCCCAACAAT TGCCAGCCAGCCCCTGAGCCTGAGGCCCGAGGCTTGTAGGCCCGCCGCTGGCGGCGCCGTCCACACCA GGGGCCTGGACTTCGCCTGCGACATCTATATCTGGGCCCCCCTGGCCGGAACCTGCGGCGTGCTGCTG CTGAGCCTGGTGATCACCCTGTACTGCAACCACAGGAACAAGAGGGGCAGGAAGAAGCTCCTGTACAT CTTCAAGCAGCCCTTCATGAGGCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCAGGTTCC CAGAGGAAGAGGAGGGCGGGTGCGAACTGAGAGTGAAATTTAGCAGGAGCGCCGACGCCCCCGCCTAT CAGCAAGGCCAGAACCAGCTGTACAACGAGCTCAACCTGGGCAGGAGGGAGGAGTACGACGTGCTGGA CAAGCGGAGGGGCAGAGATCCCGAGATGGGCGGCAAGCCCAGGAGGAAGAATCCCCAGGAGGGCCTGT ACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGGGAGAGGAGG AGGGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCT GCACATGCAGGCCCTGCCCCCCAGGConstruct 5 Amino MALPVTALLLPLALLLHAARPQVQLVQSGAEVKK 38 Acid SequencePGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEW IGATYRGHSDTYYNQKFKGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGT LVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGK APKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRFV PVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGGGGSNVLMVHKRSHTGERPFQCNQCGASFTQ KGNLLRHIKLHTGEKPFKDPGDTASAEARHIKAE MGConstruct 5 DNA ATGGCTCTTCCTGTAACCGCACTTCTGCTTCCTC 39 SequenceTTGCTCTGCTGCTTCATGCTGCTAGACCTCAGGT GCAGTTAGTGCAATCTGGAGCTGAGGTGAAGAAACCTGGCTCTTCCGTGAAAGTGAGCTGTAAGGGAA GCGGCTACACCTTTACCAACTACTGGATGCATTGGGTGAGACAGGCCCCTGGACAGGGATTAGAGTGG ATTGGAGCCACATATAGAGGACACAGCGATACCTACTACAACCAGAAGTTCAAGGGCAGGGCCACCCT TACAGCCGATACAAGCACATCTACCGCCTACATGGAACTGTCTTCTCTGAGAAGCGAGGATACCGCCG TGTACTACTGCACAAGAGGAGCCATCTACGACGGCTATGATGTTCTGGACAATTGGGGACAGGGCACA CTGGTGACAGTGTCTTCTGGTGGTGGCGGGTCCGGTGGAGGCGGATCTGGCGGTGGGGGCTCCGGAGG AGGAGGTTCAGATATTCAAATGACACAGAGCCCAAGCAGCCTGTCTGCTTCTGTGGGCGATAGAGTGA CCATCACCTGTTCTGCTTCTCAGGATATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAA GCCCCTAAACTGCTGATCTACTACACCAGCAATCTGCACTCTGGAGTTCCTAGCAGATTCAGCGGAAG CGGCTCTGGCACCGATTTTACACTGACCATCTCTTCTCTGCAGCCTGAGGATTTTGCCACCTACTACT GCCAGCAGTACCGGAAATTGCCTTGGACCTTTGGACAGGGAACCAAGCTGGAGATCAAGAGGTTTGTG CCCGTGTTTCTGCCTGCTAAGCCTACAACAACACCTGCCCCTAGACCACCTACACCTGCTCCTACAAT TGCCTCTCAGCCTCTTTCTCTGAGACCTGAAGCTTGCAGACCTGCTGCTGGAGGAGCTGTGCATACAA GAGGACTGGATTTTGCCTGCGATATCTACATTTGGGCTCCACTGGCCGGCACATGTGGAGTTCTTCTG CTGTCTCTGGTGATCACCCTGTACTGTAATCACAGGAACAAGCGGGGCCGGAAAAAGCTGCTGTACAT CTTCAAGCAGCCCTTCATGAGACCAGTTCAGACAACACAGGAGGAGGACGGCTGTAGCTGCAGATTTC CTGAGGAAGAGGAAGGAGGATGTGAATTAGGTGGTGGCGGGAGCAGGGTGAAGTTCTCACGCAGCGCA GATGCTCCTGCCTATCAGCAAGGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGAAGAGAGGA GTACGATGTGCTGGACAAGAGAAGGGGCAGAGATCCTGAAATGGGAGGAAAGCCCAGAAGGAAGAACC CTCAAGAAGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATTGGCATG AAAGGAGAGAGGAGAAGAGGAAAGGGCCATGATGGCCTGTATCAGGGCCTGTCTACAGCCACCAAGGA TACATATGATGCCCTGCATATGCAGGCTTTACCCCCTAGAGGAGGAGGCGGATCTAACGTGCTGATGG TGCATAAAAGAAGCCACACAGGAGAGAGACCATTCCAGTGCAACCAGTGTGGAGCCAGCTTCACCCAG AAGGGAAATCTGCTGAGACACATCAAACTGCATACAGGCGAGAAGCCCTTCAAGGACCCTGGCGATAC AGCCTCTGCTGAAGCTAGACACATTAAAGCCGAAATGGGC

1. A method of controlling the level of a polypeptide sequencecomprising: a) administering a fusion protein comprising saidpolypeptide sequence and a ubiquitin targeting protein consisting ofless than 135 amino acids in length which comprises the hairpin motif ofa cereblon binding site, and b) controlling the level of the polypeptidesequence by administering a compound which mediates binding of theubiquitin targeting protein and cereblon.
 2. The method of claim 1,wherein the hairpin motif comprises a sequence selected from the groupconsisting of: SEQ ID NOs: 2-5 or a functional variant thereof, whereinone or two amino acids may be substituted, added or deleted except forthe GLY residue present in each amino acid sequence.
 3. The method ofclaim 1, wherein the hairpin motif comprises a sequence selected fromthe group consisting of: SEQ ID NOs: 2-5.
 4. The method of claim 1,wherein the ubiquitin targeting protein is a polypeptide sequenceconsisting of less than 100 amino acids in length.
 5. The method ofclaim 1, wherein the ubiquitin targeting protein comprises a lysineresidue which acts as a ubiquitination site.
 6. The method of claim 1,wherein the ubiquitin targeting protein comprises a sequence selectedfrom the group consisting of: SEQ ID NOs: 6-14 and
 27. 7. The method ofclaim 1, wherein the polypeptide sequence controlled by administering acompound which mediates binding of the ubiquitin targeting protein andcereblon is a transmembrane protein.
 8. The method of claim 1, whereinthe polypeptide sequence controlled by administering a compound whichmediates binding of the ubiquitin targeting protein and cereblon is achimeric antigen receptor (CAR).
 9. The method of claim 1, wherein thecompound is an immunomodulatory imide drug (IMiD).
 10. The method ofclaim 9, wherein the IMiD is selected from thalidomide, lenalidomide,pomalidomide or a functional derivative or analog thereof.
 11. Achimeric antigen receptor (CAR) comprising: an extracellular ligandbinding domain; a transmembrane domain; an intracellular signallingdomain; and, a ubiquitin targeting protein consisting of less than 135amino acids in length which comprises the hairpin motif of a cereblonbinding site, which is capable of being bound by ubiquitin ligase in thepresence of a compound which mediates binding of the ubiquitin targetingprotein and cereblon.
 12. A fusion protein comprising a polypeptidesequence and a ubiquitin targeting protein consisting of less than 135amino acids in length which comprises the hairpin motif of a cereblonbinding site.
 13. An isolated polynucleotide encoding the ubiquitintargeting protein of claim
 1. 14. An expression vector comprising apolynucleotide of claim
 13. 15. A cell comprising a polynucleotide ofclaim
 13. 16. A cell of claim 15, which is an immunomodulatory cell. 17.A cell according to claim 16, which is a T-cell.
 18. A cell of claim 15,for use in therapy.
 19. A pharmaceutical composition comprising aplurality of cells of claim
 15. 20. The pharmaceutical composition ofclaim 19, which additionally comprises a pharmaceutically acceptableexcipient, carrier, or diluent.
 21. The pharmaceutical composition ofclaim 19, for use in therapy.
 22. The pharmaceutical composition ofclaim 21, wherein the therapy is a method of gene therapy.
 23. A methodof engineering an immunomodulatory cell, comprising: (a) providing animmunomodulatory cell; (b) transducing or transfecting thepolynucleotide of claim 13 into said immunomodulatory cell; and (c)expressing said polynucleotide in the immunomodulatory cell.